P2Y receptor-mediated inhibition of tumor necrosis factor α-stimulated stress-activated protein kinase activity in EAhy926 endothelial cells

Abstract

In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor α (TNFα)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-2, the downstream target of p38 MAP kinase. In NCT2544 keratinocytes, UTP and a proteinase-activated receptor-2 agonist caused similar inhibition, but in 13121N1 cells, transfected with the human P2Y2 or P2Y4 receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P2Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca2+ levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFα-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318220 and Go6983 reversed the inhibitory action of UTP, but GF109203X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNFα-stimulated SAP kinase pathways in endothelial cells, mediated by Ca2+-independent isoforms of protein kinase C.