Abstract
In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor alpha (TNFalpha)- 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 P2Y(2) or P2Y(4) 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 Ca(2+) levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFalpha-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 TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(2+)-independent isoforms of protein kinase C.
Highlights
In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor ␣ (TNF␣)- 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
These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNF␣-stimulated stress-activated protein (SAP) kinase pathways in endothelial cells, mediated by Ca2؉-independent isoforms of protein kinase C
In this study we examined the effect of P2Y receptor stimulation upon tumor necrosis factor ␣ (TNF␣)-stimulated SAP kinase activity in endothelial cells
Summary
Materials—The plasmid containing the cDNA encoding the GSTtagged truncated N terminus of c-Jun (GST-c-Jun5–89) was donated by J. Cohen (Medical Research Council Protein Phosphorylation Unit, University of Dundee, Scotland). We thank these people for their gifts. For immunoprecipitation of MEK-4, equal amounts of precleared cell lysates were incubated in Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 1% (w/v) Triton X-100, and 0.5% (w/v) Nonidet P-40 and with 1 g/ml MEK-4 antibody, precoupled to protein G, for 3 h. JNK and p38 MAP Kinase Activity Assay—To measure JNK activity, cells were stimulated as appropriate and the reaction terminated by rapid aspiration and the addition of ice-cold PBS. MAPKAP Kinase-2 Activity—MAPKAP kinase-2 activity in immunoprecipitates was determined by measuring incorporation of [␥-32P]phosphate into the substrate peptide KKLNRTLSVA as outlined previously [26]. Data files were saved in TIF format and analyzed using MetaMorph software (Universal Imaging, West Chester, PA)
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