P2Y(2) receptor-mediated proliferation of C-6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway

1 Extracellular purine and pyrimidine nucleotides have been implicated in t he regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C-6 g lioma cells responsible for stimulating cell proliferation associated with mitogen-activated protein kinase (MAPK) activation. 2 UTP and ATP produced a similar effect on [H-3]-thymidine incorporation in a time- and concentration-dependent manner, suggesting the involvement of P2Y(2) receptor in mediating proliferation of C-6 glioma cells. 3 In response to UTP, both p42 and p44 MAPK were activated in a time- and c oncentration-dependent manner using Western blot analysis with an anti-phos pho-p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min. 4 Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP wer e attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, pro tein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. 5 UTP-induced [H-3]-thymidine incorporation and p42/p44 MAPK phosphorylatio n was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore , we showed that overexpression of dominant negative mutants of Ras (RasN17 ) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activatio n induced by ATP and UTP. 6 These results conclude that the mitogenic effect of UTP is mediated throu gh a P2Y(2) receptor that involves the activation of Ras/Raf/MEK/MAPK pathw ay. UTP-mediated MAPK activation was modulated by Ca2+, PKC, and tyrosine k inase associated with cell proliferation in cultured C-6 glioma cells.