P2Y Receptors Activate MAPK/ERK Through a Pathway Involving PI3K/PDK1/PKC-ζ in Human Vein Endothelial Cells

Abstract

Aims: In this study we investigated the effects of P2receptors in the regulation of mitogen-activatedprotein kinase/extracellular signal-regulated kinase(MAPK/ERK) in human umbilical vein endothelial cells(HUVEC). Methods: Cytosolic Ca²⁺ concentration([Ca²⁺]_i) was measured using fura-2/AM, and MAPK/ERK phosphorylation using Western blot analysis.Results: ATP, 2-meSATP, UTP and UDP cause a rapidand transitory increase in the phosphorylation ofMAPK/ERK. In contrast, negligible response was seenfor a,ß-meATP, a general P2X receptors agonist. ATPdependentactivation of MAPK/ERK was preventedby pretreatment of HUVEC with pertussis toxin or MEKinhibitor PD98059. In addition, activation of the MAPK/ERK cascade by ATP was blocked in cells pretreatedwith wortmannin and LY294002, but not by U73122,BAPTA or a Ca²⁺-free medium. Furthermore, aninhibition of ATP-dependent MAPK/ERKphosphorylation was observed in HUVEC pretreatedwith high doses of GF109203X or myristoylated PKC-ζ pseudosubstrate. Similar results were observedwhen cells were pretreated with the Src tyrosine kinaseinhibitor PP2. However, ATP-stimulated MAPK/ERKactivation was unaffected in cells pretreated withAG1478 or perillic acid. We also found that ATPstimulates both the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1)and its translocation to plasma membrane in a timedependentmanner. Conclusion: These observationssuggest that the effects mediated by ATP in HUVECoccur via PTX-sensitive G-protein-coupled P2Yreceptors through PI3K-dependent mechanisms, inwhich PDK1 and PKC-ζ are two key molecules withinsignal cascade leading to MAPK/ERK activation.