The JAK-STAT Pathway and the Development of Cardiovascular Disease 

Despite improvements in preventative treatments, cardiovascular disease (CVD) such as congestive heart disease and stroke still claim over 130,000 lives each year in the UK. Even patients on optimal treatment regimens still have a 70-80% chance of having additional major adverse cardiovascular events, and so new strategies to combat the atherosclerosis responsible for congestive heart disease and stroke are urgently needed.  It is now known that localised inflammation of large blood vessels is a pivotal event in the initiation of atherosclerotic plaque formation. Inflammation is driven by chronic exposure to a diverse range of chemical signals termed “cytokines” which bind to receptors on vascular cells and trigger multiple intracellular pathways which ultimately lead to the defective vascular cell function responsible for cardiovascular disease. My laboratory focuses on cytokines that activate a particularly important signalling pathway in vascular endothelial and smooth muscle cells termed the “JAK-STAT” pathway. We are particularly interested in the processes that turn this pathway off, since its chronic activation in blood vessels is a key determinant of disease. However, relatively little is known about the inhibitory mechanisms responsible for turning off JAK-STAT activation in vascular cells and nothing is known regarding their status in blood vessels from patients with CVD. By expanding our knowledge of the molecular processes by which JAK-STAT signalling can be turned off, we hope to identify new targets for optimal drug treatment of affected individuals.

Inhibition of IL-6 signalling in vascular endothelial cells by cyclic AMP: Epac1-mediated induction of SOCS-3

The prototypical intracellular messenger cyclic AMP is a powerful protective molecule in vascular cells. As well as inhibiting smooth muscle cell proliferation and migration, cyclic AMP also suppresses endothelial inflammation and enhances barrier function to limit vascular permeability. Cyclic AMP classically mediated its intracellular effects by binding and activating cyclic AMP-dependent protein kinase (PKA) which phosphorylated multiple intracellular substrates to modify their activity. However, we have reported that cyclic AMP-mobilising hormones, such as adenosine and prostaglandin E2, inhibit IL-6 and leptin signalling in vascular endothelial cells via two PKA-independent routes [Sands et al., 2006]. One route involves a novel intracellular sensor termed “exchange protein activated by cyclic AMP 1” (Epac1). Upon binding cyclic AMP, Epac1 acts as a guanine nucleotide exchange factor for Rap family small G-proteins, Accumulation of Rap-GTP activates a complex signalling cascade resulting in the activation of specific protein kinase C (PKC) isoforms [Borland et al., 2009; Figure 1]. The other route triggers activation of the ERK.MAP kinase pathway [Woolson et al., 2009]. Both of these converge on a transcription factor complex containing CCAAT/enhancer binding proteins (C/EBPs) β and δ. [Yarwood et al., 2008]. The complex is recruited to the promoter of the gene encoding “suppressor of cytokine signalling 3” (SOCS-3), which then binds to and inhibits pro-inflammatory signal generation from the IL-6 receptor gp130 and the leptin receptor.

SOCS-3-mediated ubiquitylation and CVD

It is becoming clear that as well as interacting with cytokine receptors, SOCS-3 has multiple intracellular roles. An important aspect of its function is as the specificity determinant of a complex that ubiquitylates intracellular proteins, thus targeting them for degradation by the proteasome. Thus, a full assessment of SOCS-3’s role in vascular EC function requires identification of its substrates. In collaboration with Dr. Bill Mullen and Dr. Richard Burchmore in the Institute of Infection, Immunity and Inflammation, we have used quantitative proteomics to compare the ubiquitinome from SOCS-3-expressing and SOCS-3-null cells to identify those proteins whose ubiquitylation status is regulated by SOCS-3. Several new proteins have been identified by this route and the biological importance of their interaction with SOCS-3 is currently being investigated. In collaboration with Prof. Andy Baker, we are also examining how inhibiting the ubiquitylation of SOCS-3 itself could be used to increase its stability and thus reduce the localized inflammation and smooth cell migration responsible for the restenosis which limits the long-term success of angioplasty and coronary artery bypass graft procedures.

Inhibition of JAK-STAT signalling by AMPK

AMPK is a serine/threonine protein kinase involved in the regulation of cellular and organismal metabolism and has been proposed to be a candidate target for therapeutic intervention in the treatment of type 2 diabetes and insulin resistance. Several studies have revealed that AMPK is required for maintenance of healthy vascular function but the molecular mechanisms responsible remain to be fully defined. In collaboration with Dr Ian Salt, we have identified a novel molecular mechanism linking AMPK to the rapid suppression of pro-inflammatory JAK-STAT-mediated signalling responses. Crucially, in collaboration with Dr Christian Delles, we have found that the ability of this pathway to inhibit JAK-STAT signalling is rapidly compromised in endothelial cells from patients with coronary artery disease. We are currently examining the molecular basis of this defect in order to devise strategies aimed at rescuing functionality in disease.

JAK-STAT signalling and the development of pulmonary hypertension

IL-6 is a key driver of the chronic inflammation observed in the pulmonary vasculature during the development of pulmonary arterial hypertension (PAH), a debilitating condition with a poor prognosis. Individuals who are homozygous for inactivating mutations within the BMPRII receptor gene are at increased risk of developing this condition. In collaboration with Prof. Mandy MacLean, we are investigating how inactivation of BMPRII signalling is mechanistically linked to the establishment of the chronic inflammation that drives the observed pulmonary vessel remodelling.

Publications

• Safhi MMA, Rutherford C, Ledent C, Sands WA, Palmer TM. Priming of signal transducer and activator of transcription proteins for polyubiquitylation and degradation by the A2A adenosine receptor. Mol Pharmacol 2010;77:968–978.
• Mundell SJ, Matharu A-L, Palmer TM, Benovic JL, Kelly E. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches agonist-induced internalisation to an arrestin- and clathrin-independent pathway and inhibits receptor recycling. Br J Pharmacol 2010;159:518–533.
• Woolson HD, Thompson VS, Rutherford C, Yarwood SJ, Palmer TM. Selective inhibition of cytokine-activated extracellular signal-regulated kinase by cyclic AMP via Epac1-dependent induction of suppressor of cytokine signalling-3. Cell Signal 2009;21:1706–1715.
• Borland G, Bird R, Palmer TM, Yarwood SJ. Activation of protein kinase C α by EPAC1 is required for the ERK- and C/EBPβ-dependent induction of the SOCS-3 gene by cyclic AMP in COS1 cells. J Biol Chem 2009;284:17391–17403.
• Yarwood SJ, Borland G, Sands WA, Palmer TM. Identification of C/EBPs as EPAC-activated transcription factors that mediate the induction of the SOCS-3 gene. J Biol Chem 2008;283(11):6843-53.
• Mair KM, MacLean MR, Morecroft I, Dempsie Y, Palmer TM. Novel interactions between the 5-HT transporter, 5-HT1B receptors and Rho kinase in vivo and in pulmonary fibroblasts. Br J Pharmacol 2008;155:606-616.
• Palmer TM, Trevethick MA. Suppression of inflammatory and immune responses by the A(2A) adenosine receptor: an introduction. Br J Pharmacol 2008;153:S27–S34.
• Sands WA, Palmer TM. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal 2008;20(3):460-6.
• Funakoshi H, Chan TO, Good JC, Libonati JR, Piuhola J, Chen X, MacDonnell SM, Lee LL, Herrmann DE, Zhang J, Martini J, Palmer TM, Sanbe A, Robbins J, Houser SR, Koch WJ, Feldman AM. Regulated overexpression of the A1-adenosine receptor in mice results in adverse but reversible changes in cardiac morphology and function. Circulation 2006;114(21):2240-50.
• Sands WA, Woolson HD, Milne GR, Rutherford C, Palmer TM. Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells. Mol Cell Biol 2006;26(17):6333-46.
• Sands WA, Palmer TM. Inhibition of pro-inflammatory cytokine receptor signalling by cAMP in vascular endothelial cells. Biochem Soc Trans 2005;33(Pt 5):1126-8.
• Rutherford C, Ord-Shrimpton FU, Sands WA, Pediani JD, Benovic JL, McGrath JC, Palmer TM. Phosphorylation-independent internalisation and desensitisation of the human sphingosine-1-phosphate receptor S1P3. Cell Signal 2005;17(8):997-1009.