Protective model of G-protein coupled receptor activation in reducing cytokine signalling (TNF-α and IL-1β) in HUVECs

Pei Yuen Ng, Kathryn McIntosh, Robin Plevin. Strathclyde Institute for Pharmacy and Biomedical Sciences (SIPBS), G4 0RE, Glasgow, United Kingdom.

In the blood vessel lumen, fluid shear stress promotes the release of ATP from the endothelial cells (Milner et al. 1990) to stimulate purinergic receptors that are expressed on the cell surface. In this study, we sought to examine the possible role of GPCR activation in protection of the endothelium by reducing inflammatory signalling. Previous studies have demonstrated that stimulation of proteinase-activated receptor-2 (PAR2) and P2Y2 reduced TNF-α mediated JNK signalling in PAR2 overexpressing cell lines and EAhy926 cells (Paul et al. 2000, McIntosh et al. 2010). Thus, this study explored the effect of GPCR activation on cytokine receptor signalling in primary cultures, namely human umbilical vein endothelial cells (HUVECs). MAPK and c-Jun phosphorylation was measured using Western blotting and JNK activity using solid phase in vitro kinase assay using GST-tagged c-Jun. Initial results demonstrated that ATP and the cytokines (TNF-α and IL-1β) strongly activated MAPKs, but NF-κB activation was only detected following cytokine stimulation. In terms of JNK signalling, 30 μM ATP activated P2Y receptors (Wang et al. 2002) to give a 3.47 ± 0.46 fold stimulation which was weaker compared to cytokines (Fold stimulation; 20 ng/ml TNF-α: 6.82 ± 0.40, 10 ng/ml IL-1β: 11.16 ± 0.97 n = 4). Nevertheless, pre-incubation of 30 μM ATP for 30 min was sufficient to reduce JNK activity mediated by both TNF-α and IL-1β by approximately half (% inhibition TNF-α = 45.50 ± 6.42, IL-1β = 35.89 ± 3.84, n = 3 respectively). These kinase assay results were confirmed with p-JNK and p-c-Jun Western blotting. The concentration-dependent inhibition of ATP on JNK signalling was pathway specific; as cytokine induced p38 MAPK, p65 NF-κB phosphorylation and IκB degradation were not significantly affected. In order to verify that this inhibition occurs in a GPCR-cytokine receptor specific manner, sorbitol was used as an alternative JNK activator. Although sorbitol at 0.5M strongly activated JNK activity, this JNK activation by sorbitol was unaffected by ATP pre-incubation. Further studies on the kinetics of GPCR-cytokine inhibition for 2 hours revealed that co-incubation of ATP with either cytokines significantly shortened the duration of JNK activity as observed at 30 mins. Overall, this data suggested that the JNK signalling inhibition phenomenon of P2Y on cytokines is observed in primary cell cultures expressing endogenous levels of P2Y receptors.

* All numerical data are expressed as mean ± standard error mean.

Milner, P., K. A. Kirkpatrick, et al. (1990). Endothelial cells cultured from human umbilical vein release ATP, substance P and acetylcholine in response to increased flow. Proc Biol Sci 241(1302): 245-8.

Paul, A., L. J. Torrie, et al. (2000). P2Y receptor-mediated inhibition of tumor necrosis factor alpha -stimulated stress-activated protein kinase activity in EAhy926 endothelial cells. J Biol Chem 275(18): 13243-9.

McIntosh, K., M. R. Cunningham, et al. (2010). Proteinase-activated receptor-2 mediated inhibition of TNF alpha-stimulated JNK activation - A novel paradigm for G(q/11) linked GPCRs. Cell Signal 22(2): 265-73.

Wang L, Karlsson L et al (2002) P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol 40(6): 841-53.

Pei Yuen NG is sponsored by an Agung scholarship from the Malaysian Public Service Department.