Reduction of cytokine receptor mediated JNK signalling via purinergic receptor activation: An anti-inflammatory role for G-protein coupled receptors

Pei Yuen Ng, Kathryn McIntosh, Robin Plevin. University of Strathclyde, 161 Cathedral St, G4 0RE, UK

Ubiquitously expressed purinergic receptors, which are activated by extracellular nucleotides, have been studied extensively to understand their role in both physiological and diseased conditions.1 On the surface of endothelial cells, P2Y receptors2 are exposed to ATP, which is released in response to fluid shear stress, and associated degradation products ADP and adenosine. Thus, we examined the effect of P2Y receptor stimulation in endothelial cells and cross talk with cytokine mediated JNK signalling. In human umbilical vein endothelial cells (HUVECs), ATP alone gave a small transient JNK signal, however pre-treatment of ATP inhibited cytokine (IL-1β) mediated JNK signalling in a concentration dependent manner (% inhibition by 100 µM ATP = 68.9 ± 12.7% and 67.4 ± 6.1%, p<0.05, by kinase assay and Western blotting respectively). Responses to sorbitol and anisomycin were not affected by ATP and the inhibition was pathway specific; p38MAP kinase and NF-κB signalling was unaltered. Pre-incubation with the P2Y11 antagonist, NF340, reversed the inhibitory effect on IL-1β mediated JNK signalling, however inhibition was unaffected in the presence of the ecto-nucleotidase inhibitor (ARL67156). We then attempted to uncover the signalling mechanisms downstream of P2Y11 that is involved in the inhibition of IL-1β mediated JNK signalling. Pre-incubation with the G protein Gq/11 inhibitor YM-254890, partially reversed the inhibitory action of ATP on IL-1β stimulated JNK (% inhibition of JNK activity: 100 μM ATP = 80.7 ± 6.4%, 100 nM YM+ ATP = 52.9 ± 13.5%). The protein kinase A inhibitor H89 also reversed the ATP inhibitory effect on IL-1β mediated JNK signalling (% inhibition of JNK activity: 30 μM ATP = 55.6 ± 5.3%, 10 μM H89+ ATP = 19.7 ± 5.1%, p< 0.001). This inhibition of JNK signalling by ATP was also translated into a physiological outcome as measured by reduction of pro-inflammatory COX-2 production by IL-1β (45.0 ± 4.4 % inhibition by 100 μM ATP). These results collectively support the emerging hypothesis that activation of GPCRs, for example P2Y11, can inhibit cytokine mediated JNK signalling via both Gq/11 and Gs linked pathways. In the context of endothelial cell function, purinergic receptors could exert an anti-inflammatory effect via these mechanisms. Thus selective, high affinity P2Y11 receptor agonists could be developed as a therapy for inflammatory mediated diseases. Future studies will focus on the mechanisms downstream of Gq/11 and Gs that inhibit cytokine receptor activation of JNK.

References:

1. Erlinge D (2011) P2Y receptors in health and disease. Adv Pharmacol, 61, 417-39.

2. Wang, L., L. Karlsson, S. Moses, A. Hultgardh-Nilsson, M. Andersson, C. Borna, T. Gudbjartsson, S. Jern & D. Erlinge (2002) P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol, 40, 841-53.