Chronic inflammatory diseases, such as atherosclerosis, are a major cause of death and disability in the developed world. In this respect, although cholesterol obviously plays a predominant role in atherosclerosis, targeting inflammation at lesion sites may be just as important. Indeed, elevated IL-6 (interleukin 6) levels are as strongly associated with coronary heart disease as increased cholesterol. We have been investigating novel cAMP-regulated pathways that combat the action of pro-inflammatory cytokines, such as IL-6 and leptin, in the VECs (vascular endothelial cells) of the circulatory system. In this respect, we have begun to unravel new molecular mechanisms by which the cAMP/Epac1 (exchange protein directly activated by cAMP 1)/Rap1 pathway can initiate a rigorous programme of protective anti-inflammatory responses in VECs. Central to this is the coupling of cAMP elevation to the mobilization of two C/EBP (CCAAT/enhancer-binding protein) family transcription factors, resulting in the induction of the SOCS3 (suppressor of cytokine signalling 3) gene, which attenuates pro-inflammatory cytokine signalling in VECs. These novel ‘protective’ mechanisms of cAMP action will inform the development of the next generation of pharmaceuticals specifically designed to combat endothelial inflammation associated with cardiovascular disease.

Endothelial dysfunction and vascular disease

The vascular endothelium forms a multifunctional anti-coagulant, anti-inflammatory and anti-thrombotic barrier to infection and injury. However, VECs (vascular endothelial cells) can dramatically alter their phenotype to evoke an inflammatory environment in response to a variety of chemical stimuli (e.g. lipids, pro-inflammatory cytokines and pathogen-derived molecules) as well as mechanical injury and hypoxia. Acquisition of this so-called ‘dysfunctional’ phenotype is a pivotal event in the pathogenesis of several debilitating diseases that include sepsis [1], allograft rejection [2] and atherosclerosis, which results in heart disease and stroke [3]. Although rapid systemic inflammation and vascular leakage characterizes the development of sepsis, heart disease and stroke are triggered by chronic vascular inflammation at specific sites within the arterial tree. This promotes excessive adhesion of monocytes and T-cells to vessel walls before their migration into the intimal layer where they initiate the smooth muscle cell proliferation that leads to atherosclerotic plaque formation, vessel occlusion and rupturing [2,3].

For diseases associated with endothelial dysfunction, molecular processes that dampen exaggerated inflammatory responses in VECs are particularly attractive targets for the development of new therapeutics because these responses are potentially reversible [4]. For example, whereas avoidance of risk factors is the preferred approach to prevent atherosclerosis, widespread patient non-compliance and the influence of genetic factors in determining individual predisposition has led to the development of preventative pharmacological strategies, with lipid-lowering ‘statins’ being the most successful. However, many individuals either display a resistance to statin monotherapy or suffer from a range of side effects [5]. In addition, it is now appreciated that, whereas antibiotics are an essential aspect of management of sepsis, adjuvant therapy using drugs that target the host response to infection will be essential if the substantial 35% mortality rate due to this condition is to be reduced [6]. Consequently, it can be seen that there is a pressing need to develop new small-molecule inhibitors of VEC dysfunction for the many conditions in which this process is a key trigger of disease pathology.

Although numerous studies have shown that activation of the transcription factor NF-κB (nuclear factor κB) in the endothelium is an early event in the development of vascular inflammation [3], several important pro-inflammatory mediators utilize alternative signalling pathways to control VEC function, including class I cytokines such as leptin and IL-6 (interleukin-6). Upon its release by activated VECs, smooth muscle cells and macrophages, IL-6 can accumulate at sites of vascular injury, and its effects on VECs include the induction of the chemokine MCP-1 (monocyte chemoattractant protein-1)/CCL2 (CC chemokine ligand 2) and VCAM-1 (vascular cell adhesion molecule 1), which both work to promote the arrest of rolling monocytes to inflamed endothelium [7,8]. Signalling by IL-6 in VECs occurs through the IL-6R (IL-6 receptor) complex, composed of an IL-6-binding α-chain (IL-6Rα) and gp130 (glycoprotein 130), which interacts with IL-6Rα [9]. Receptor clustering takes place following the binding of the complex to gp130, leading to the activation of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. Activated STAT3 homodimerizes and translocates to the nucleus, where it acts as a transcription factor for the induction of pro-inflammatory IL-6-responsive genes [10]. Clearly, regulation of such pro-inflammatory signalling is vital to prevent runaway inflammation. One crucial mechanism for down-regulating JAK/STAT signalling is therefore via the SOCS (suppressor of cytokine signalling) family of proteins [11]. For example, SOCS3 binds to JAK-phosphorylated receptors, via the SOCS3 SH2 (Src homology 2) domain, thereby inhibiting JAK activity and, consequently, activating STATs 1 and 3 [12]. SOCS3 then also targets SH2-bound proteins for proteasomal degradation [12]. SOCS proteins are often induced directly by the same JAK/STAT pathway that they inhibit, forming a classical negative-feedback loop.

We demonstrated recently that elevations in the levels of intracellular cAMP levels, triggered by activation of adenosine and prostaglandin receptors, in HUVECs (human umbilical vein endothelial cells), leads to inhibition of signalling from the IL-6–soluble IL-6Rα trans-signalling complex to phosphorylation and activation of STAT3 and the MAPK (mitogen-activated protein kinase) ERK (extracellular-signal-regulated kinase) [10]. This inhibition was found to be independent of the classical route for cAMP signalling, through PKA (protein kinase A), but rather dependent on induction of the SOCS3 gene in response to activation of Epac (exchange protein directly activated by cAMP) 1 [10]. Manipulation of intracellular cAMP levels can therefore be used to subvert the usual JAK/STAT negative-feedback loop in order to produce a blockade of cytokine signalling in VECs. Given that manipulation of cellular cAMP levels currently forms the basis of many effective pharmaceuticals [13], e.g. Ariflo (Cilomilast) for the treatment of COPD (chronic obstructive pulmonary disorder) and forskolin analogues used for the treatment of asthma in Japan, cAMP-regulated signalling pathways represent an attractive therapeutic target for limiting endothelial dysfunction associated with atherosclerosis and other diseases of the cardiovascular system.

Epac1 is a central controller of anti-inflammatory processes in VECs

We have begun to delineate the molecular basis of how elevations in intracellular cAMP positively control the expression of the SOCS3 gene and thereby inhibit pro-inflammatory cytokine signalling in VECs. In this respect, we have found that activation of Epac1 is absolutely required for SOCS3 induction by cAMP in a variety of cell types, including HUVECs [10,14]. The Epacs, Epac1 and Epac2, are specific GEFs (guanine-nucleotide-exchange factors) for the Ras GTPase homologues Rap1 and Rap2, which they activate independently of PKA [15]. Rather, cAMP-binding sites in Epacs facilitate their direct activation by cAMP, thereby relieving autoinhibitory influences of the cAMP-binding domain towards the catalytic GEF domain [15]. It is now becoming clear that Epac1 mediates at least three cAMP-activated anti-inflammatory signalling pathways in VECs (Figure 1): (i) down-regulation of IL-6-mediated inflammatory processes [10], which occurs through C/EBP (CCAAT/enhancer-binding protein) transcription factor-dependent SOCS3 induction [14], (ii) limiting vascular permeability through Epac1-mediated activation of integrins involved in the adhesion of VECs to the basement membrane [16], and (iii) promotion of endothelial barrier function through actin- [1721] and microtubule- [22] dependent cell–cell junction formation through stabilization of VE-cadherin (vascular endothelial cadherin)-mediated adhesion [23]. Overall, the involvement of Epac1 in the control of multiple anti-inflammatory mechanisms in VECs presents an intriguing model in which to study how distinct cellular processes may interact to present a co-ordinated programme of ‘protection’ against inflammatory stimuli (Figure 1). Clearly, further delineation of the molecular mechanisms connecting cAMP and Epac1 to this protective control network would be anticipated to make a significant contribution to the development of the next generation of anti-atherogenic pharmaceuticals designed to limit the action of inflammatory stimuli in VECs.

Anti-inflammatory effects of Epac1 in VECs

Figure 1
Anti-inflammatory effects of Epac1 in VECs

Epac1 activation, in response to G-protein-coupled receptor (GPCR)-stimulated adenylate cyclase (AC)-mediated cAMP production, mediates at least three anti-inflammatory signalling pathways in VECs. (i) IL-6 signalling through JAK/STATs is inhibited by Epac1-dependent SOCS3 induction; this occurs via Rap1 and C/EBP transcription factors. (ii) Epac mediates activation of integrins involved in adhesion of VECs to the basement membrane. (iii) Epac reduces endothelial permeability by inhibiting Rho activation and actin cytoskeleton reorganization, by promoting microtubule lengthening and by unidentified signalling pathways not requiring Rho or the cytoskeleton, all of which result in increased VE-cadherin-mediated adhesion.

Figure 1
Anti-inflammatory effects of Epac1 in VECs

Epac1 activation, in response to G-protein-coupled receptor (GPCR)-stimulated adenylate cyclase (AC)-mediated cAMP production, mediates at least three anti-inflammatory signalling pathways in VECs. (i) IL-6 signalling through JAK/STATs is inhibited by Epac1-dependent SOCS3 induction; this occurs via Rap1 and C/EBP transcription factors. (ii) Epac mediates activation of integrins involved in adhesion of VECs to the basement membrane. (iii) Epac reduces endothelial permeability by inhibiting Rho activation and actin cytoskeleton reorganization, by promoting microtubule lengthening and by unidentified signalling pathways not requiring Rho or the cytoskeleton, all of which result in increased VE-cadherin-mediated adhesion.

Transcriptional regulation of the SOCS3 gene by novel signalling through Epac1

In an effort to understand the link between Epac1 activation and SOCS3 induction in VECs, we have been concentrating our efforts on identifying downstream targets of Epac1 and Rap1. Recent reports had already begun to link Epac1 activation to Rap-dependent stimulation of PLC (phospholipase C) ϵ [23,24], raising the possibility that classical downstream mediators of PLC signalling, such as PKC (protein kinase C), may be targets of Epac1 signalling in VECs. Indeed, there has been some suggestion that in neurons and heart, at least, activation of PKC, particularly PKCϵ, by Epac may mediate responses such as pain and inflammation [2528]. Consistent with this was our discovery that the PKC isoforms α and δ are a critical requirement for efficient SOCS3 induction by cAMP and Epac1 in COS1 cells [29] and HUVECs (J. Wiejak and S.J. Yarwood, unpublished work). The mechanisms linking PKC to Epac1 and Rap1 activation appears to involve a pathway including PLCϵ, Ca2+ and DAG (diacylglycerol) [29].

Downstream signalling from cAMP-activated PKC isoforms probably involves the C/EBP transcription factors C/EBPβ and C/EBPδ, which interact directly with the SOCS3 promoter and are necessary and sufficient for the induction of the SOCS3 gene in HUVECs [14]. The six C/EBP proteins (α, β, γ, δ, ϵ and ζ) are structurally similar bZIP (basic leucine zipper) transcription factors that appear to function as master regulators of diverse cellular processes such as differentiation and inflammatory responses [30]. Importantly, C/EBP transactivation is thought to be regulated directly by cAMP [31,32]. Indeed, studies have demonstrated that the cAMP-responsive domains on C/EBP lack PKA phosphorylation sites [31,32]. This is evidence that C/EBPs can preferentially induce transcription in response to cAMP in a PKA-independent fashion and may well involve Epac1, as our results suggest [14].

The mechanism by which Epac1 activates C/EBP transcription factors remains unclear, but probably depends on phosphorylation of C/EBP proteins by intermediate cAMP-activated protein kinases. Indeed, it has been demonstrated that certain C/EBP isoforms are substrates for ERK, RSK (ribosomal S6 kinase) and PKC protein kinases [33]. In this respect, we have found that C/EBPβ, but not C/EBPδ, is phosphorylated in cells response to cAMP elevation and PKC activation (Figure 2). Moreover, using a combination of phospho-specific antibodies and mutation of C/EBPβ, we have found that SOCS3 induction, in response to elevations in intracellular cAMP levels, requires the ERK-dependent phosphorylation of C/EBPβ on Thr235 [29,34] (Figure 3). An outstanding question arising from these studies, however, is how elevations in intracellular cAMP lead to the activation of the ERK cascade [29]. It has been known for some time that cAMP can trigger ERK activation in a wide variety of immortalized and primary cultured cells, including pre-adipocytes [35], neuronal-derived PC12 cells [36] and brown adipocytes [37]; however, the mechanisms by which this occurs remain elusive to date. We know that in HUVECs, at least, the contribution of Epac1 to ERK activation is minimal and that PKA activity is completely dispensable [34,38]. We have also found that cAMP elevation leads to Ras activation in these cells (S.J. Yarwood, unpublished work). It is possible therefore that a cAMP-activated Ras GEF may be involved; for example, we know that the cAMP-regulated Ras/Rap1 GEF CNRasGEF or RapGEF2 [39] is expressed in HUVECs, (S.J. Yarwood, unpublished work) and has been reported to mediate the actions of cAMP on ERK activity in human melanoma cells [40]. However, whether or not CNRasGEF plays a role in regulating anti-inflammatory signalling in VECs remains to be formally tested.

C/EBPβ, but not C/EBPδ, is phosphorylated in response to elevation of cAMP or activation of PKC

Figure 2
C/EBPβ, but not C/EBPδ, is phosphorylated in response to elevation of cAMP or activation of PKC

COS1 cells were transfected with FLAG-tagged human C/EBPα, C/EBPβ and C/EBPδ. Cells were then incubated overnight with [32P]Pi and then treated for 1 h with either 10 μM PMA, to activate conventional and novel PKC isoforms, or with a combination (F/R) of the adenylate cyclase activator forskolin (10 μM) and the cAMP-phosphodiesterase inhibitor rolipram (10 μM), to elevate intracellular cAMP levels. C/EBPs were then immunoprecipitated from cell extracts with anti-FLAG antibodies and the 32P-labelled proteins were visualized by autoradiography following SDS/PAGE. The upper arrow indicates phosphorylated C/EBPβ and the lower arrow indicates phosphorylated C/EBPδ.

Figure 2
C/EBPβ, but not C/EBPδ, is phosphorylated in response to elevation of cAMP or activation of PKC

COS1 cells were transfected with FLAG-tagged human C/EBPα, C/EBPβ and C/EBPδ. Cells were then incubated overnight with [32P]Pi and then treated for 1 h with either 10 μM PMA, to activate conventional and novel PKC isoforms, or with a combination (F/R) of the adenylate cyclase activator forskolin (10 μM) and the cAMP-phosphodiesterase inhibitor rolipram (10 μM), to elevate intracellular cAMP levels. C/EBPs were then immunoprecipitated from cell extracts with anti-FLAG antibodies and the 32P-labelled proteins were visualized by autoradiography following SDS/PAGE. The upper arrow indicates phosphorylated C/EBPβ and the lower arrow indicates phosphorylated C/EBPδ.

C/EBP-dependent induction of the SOCS3 gene in VECs occurs through parallel cAMP-activated signalling pathways

Figure 3
C/EBP-dependent induction of the SOCS3 gene in VECs occurs through parallel cAMP-activated signalling pathways

cAMP elevation following stimulation of adenosine or prostaglandin receptors in VECs leads to activation of the small GTPases Rap1 and Ras, independently of the classical PKA route of cAMP signalling. Ras then activates the ERK cascade, leading to phosphorylation and activation of C/EBPβ transcription factor [14,29,34]. Rap1 activation by Epac1 leads to activation of C/EBPs β and δ [14], through a route parallel to the Ras/ERK cascade, involving activation of PLCϵ and PKC isoform α [29]. Inherent to this scheme is the potential for cross-talk between activated PKCα and the Raf-1/ERK pathway. DAG, diacylglycerol; MEK, MAPK/ERK kinase.

Figure 3
C/EBP-dependent induction of the SOCS3 gene in VECs occurs through parallel cAMP-activated signalling pathways

cAMP elevation following stimulation of adenosine or prostaglandin receptors in VECs leads to activation of the small GTPases Rap1 and Ras, independently of the classical PKA route of cAMP signalling. Ras then activates the ERK cascade, leading to phosphorylation and activation of C/EBPβ transcription factor [14,29,34]. Rap1 activation by Epac1 leads to activation of C/EBPs β and δ [14], through a route parallel to the Ras/ERK cascade, involving activation of PLCϵ and PKC isoform α [29]. Inherent to this scheme is the potential for cross-talk between activated PKCα and the Raf-1/ERK pathway. DAG, diacylglycerol; MEK, MAPK/ERK kinase.

Together, these findings reveal a central co-ordinated role for Epac1 and the ERK MAPK cascade in governing novel gene regulation involving cross-talk between the cAMP and PKC signalling pathways. Central to this scheme is the idea that C/EBPβ and C/EBPδ are activated by separate cAMP-regulated signalling pathways that converge on the induction of the SOCS3 gene, perhaps through co-operative dimerization [30] (Figure 3). Work from an increasing number of laboratories, including our own, has begun to implicate Epac1 as a ‘master regulator’ of protective anti-inflammatory mechanisms in VECs and may therefore serve to limit the damaging actions of pro-inflammatory cytokines associated with cardiovascular diseases such as atherosclerosis. We have begun to delineate the intracellular mechanisms linking Epac1 activation to the induction of the anti-inflammatory SOCS3 gene and hypothesize therefore that targeted activation of this pathway may limit endothelial dysfunction associated with cardiovascular disease. Indeed, Epac1 itself may prove to be an effective therapeutic target given that it can be selectively activated by both Epac-specific cAMP analogues [41] and through potential allosteric mechanisms as manifested by anti-diabetic sulfonylurea drugs [42].

Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).

Abbreviations

     
  • C/EBP

    CCAAT/enhancer-binding protein

  •  
  • Epac

    exchange protein directly activated by cAMP

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • GEF

    guanine-nucleotide-exchange factor

  •  
  • gp130

    glycoprotein 130

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • IL-6

    interleukin-6

  •  
  • IL-6R

    IL-6 receptor

  •  
  • JAK

    Janus kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • PKA

    protein kinase A

  •  
  • PKC

    protein kinase C

  •  
  • PLC

    phospholipase C

  •  
  • SH2

    Src homology 2

  •  
  • SOCS

    suppressor of cytokine signalling

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • VEC

    vascular endothelial cell

  •  
  • VE-cadherin

    vascular endothelial cadherin

Funding

S.J.Y. and T.M.P. are funded by project grants from the British Heart Foundation [grant numbers PG/10/026/28303 (to S.J.Y.) and PG/08/125 (to T.M.P.)].

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