Investigation of the cAMP-mediated inhibitory mechanism on the signalling pathways of 2 cytokines: IL-6 and leptin in endothelial cells

There is a wealth of evidence to support the anti-inflammatory properties of the prototypical second messenger cyclic-AMP (cAMP), notably with regard to endothelial function. Many studies have shown that cAMP can limit vascular permeability by enhancing barrier function and reducing pro-inflammatory effects of cytokines. Although the protective effects of cAMP elevation on limiting endothelial dysfunction have been well documented, the exact molecular mechanisms remain unclear. Using two endothelial cell types, namely human umbilical vein endothelial cells (HUVECs) and a novel human endothelial angiosarcoma-derived cell line (AS-M), this study has further characterised the cAMP-mediated inhibitory mechanism on the signalling pathways of two cytokines; interleukin-6 (IL-6) and leptin. Both cytokines have been implicated in the regulation of the immune response and both have been shown to play important pathological roles in various inflammatory diseases. In preliminary studies, cAMP elevation was shown to induce suppressor of cytokine signalling 3 (SOCS3) in HUVECs. Further investigation of this SOCS protein in the context of IL-6 and leptin signalling in endothelial cells would be of interest in terms of possibly elucidating the molecular mechanisms underlying the protective effects of cAMP. Results from this study demonstrated a cAMP-mediated inhibition of soluble IL-6Rα (sIL-6R)/IL-6-stimulated extracellular regulated mitogen-activated protein kinase 1, 2 (ERK1,2) and signal transducer and activator of transcription 3 (STAT3) activation in HUVECs, which was independent of cAMP-dependent protein kinase A (PKA). Instead, results demonstrated the involvement of the other major cAMP sensor; exchange protein activated by cAMP 1 (Epac1). Moreover, this inhibition was shown to be SOCS3-dependent. There also appeared to be a requirement for ERK1,2 activation in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated STAT3 activation in HUVECs. In contrast to these findings, cAMP-mediated inhibition of leptin-stimulated STAT3 activation in HUVECs was shown to occur via a SOCS3-independent mechanism. The responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated ERK1,2 activation in AS-Ms were variable, since basal levels of ERK1,2 activation were high. Furthermore, the responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated STAT3 activation in AS-Ms were either very modest or showed no effect, respectively. SOCS3 was not shown to be involved in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated ERK1,2 and STAT3 activation in AS-Ms. In conclusion, this study further characterised the cAMP-mediated inhibitory mechanism in HUVECs and AS-Ms, with a particular focus on the ERK1,2 signalling pathway of IL-6 and leptin. Despite varying results between both cell types, this study also identified AS-Ms as a useful and tractable cell model to study in the context of endothelial biology. Thus, a potentially new pathway has been identified which inhibits cytokine receptor activation of ERK1,2 and STAT3 in endothelial cells. A better understanding of this mechanism could contribute towards new therapeutics in the area of chronic inflammatory diseases, such as atheroscleriosis

Investigation of the cAMP-mediated inhibitory mechanism on the signalling pathways of 2 cytokines : IL-6 and leptin in endothelial cells

There is a wealth of evidence to support the anti-inflammatory properties of the prototypical second messenger cyclic-AMP (cAMP), notably with regard to endothelial function. Many studies have shown that cAMP can limit vascular permeability by enhancing barrier function and reducing pro-inflammatory effects of cytokines. Although the protective effects of cAMP elevation on limiting endothelial dysfunction have been well documented, the exact molecular mechanisms remain unclear. Using two endothelial cell types, namely human umbilical vein endothelial cells (HUVECs) and a novel human endothelial angiosarcoma-derived cell line (AS-M), this study has further characterised the cAMP-mediated inhibitory mechanism on the signalling pathways of two cytokines; interleukin-6 (IL-6) and leptin. Both cytokines have been implicated in the regulation of the immune response and both have been shown to play important pathological roles in various inflammatory diseases. In preliminary studies, cAMP elevation was shown to induce suppressor of cytokine signalling 3 (SOCS3) in HUVECs. Further investigation of this SOCS protein in the context of IL-6 and leptin signalling in endothelial cells would be of interest in terms of possibly elucidating the molecular mechanisms underlying the protective effects of cAMP. Results from this study demonstrated a cAMP-mediated inhibition of soluble IL-6Rα (sIL-6R)/IL-6-stimulated extracellular regulated mitogen-activated protein kinase 1, 2 (ERK1,2) and signal transducer and activator of transcription 3 (STAT3) activation in HUVECs, which was independent of cAMP-dependent protein kinase A (PKA). Instead, results demonstrated the involvement of the other major cAMP sensor; exchange protein activated by cAMP 1 (Epac1). Moreover, this inhibition was shown to be SOCS3-dependent. There also appeared to be a requirement for ERK1,2 activation in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated STAT3 activation in HUVECs. In contrast to these findings, cAMP-mediated inhibition of leptin-stimulated STAT3 activation in HUVECs was shown to occur via a SOCS3-independent mechanism. The responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated ERK1,2 activation in AS-Ms were variable, since basal levels of ERK1,2 activation were high. Furthermore, the responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated STAT3 activation in AS-Ms were either very modest or showed no effect, respectively. SOCS3 was not shown to be involved in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated ERK1,2 and STAT3 activation in AS-Ms. In conclusion, this study further characterised the cAMP-mediated inhibitory mechanism in HUVECs and AS-Ms, with a particular focus on the ERK1,2 signalling pathway of IL-6 and leptin. Despite varying results between both cell types, this study also identified AS-Ms as a useful and tractable cell model to study in the context of endothelial biology. Thus, a potentially new pathway has been identified which inhibits cytokine receptor activation of ERK1,2 and STAT3 in endothelial cells. A better understanding of this mechanism could contribute towards new therapeutics in the area of chronic inflammatory diseases, such as atheroscleriosis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Exchange protein directly activated by cyclic AMP-1-regulated recruitment of CCAAT/enhancer-binding proteins to the suppressor of cytokine signaling-3 promoter

The ability of prototypical second messenger cyclic AMP (cAMP) to positively control transcription of the somatostatin gene was pivotal to the original identification of the transcription factor cAMP response element-binding protein. However, it is now clear that alternative intracellular cAMP sensors, of which the exchange protein directly activated by cAMP (Epac) proteins have been studied most intensively, also initiate transcription of key genes in response to cAMP elevation. For example, we have demonstrated in vascular endothelial cells that activation of Epac1 is necessary for cAMP-mobilizing agents to trigger the induction of the gene-encoding suppressor of cytokine signaling-3 (SOCS-3), a potent inhibitor of interleukin (IL)-6 signaling. This is achieved through the recruitment of CCAAT/enhancer-binding protein (C/EBP) transcription factors to the SOCS-3 promoter. Here, we describe in detail how to identify and measure cAMP-mediated recruitment of a specific C/EBP isoform to a candidate regulator region of the SOCS-3 promoter in vascular endothelial cells in vitro. We also describe the RNA interference strategies with which we identified a role for Epac1 and SOCS-3 in being responsible for mediating the inhibitory effect of cAMP elevation on IL-6 signaling

Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Rα/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects

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

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-&#945;/IL-6 (sIL-6R-&#945;/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/EBP&#946;, 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 fibroblas