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 the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C(6) glioma cells responsible for stimulating cell proliferation associated with mitogen-activated protein kinase (MAPK) activation. 2. UTP and ATP produced a similar effect on [(3)H]-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 concentration-dependent manner using Western blot analysis with an anti-phospho-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 were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. 5. UTP-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation 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 activation induced by ATP and UTP. 6. These results conclude that the mitogenic effect of UTP mediated through a P2Y(2) receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in cultured C(6) glioma cells.
Tu
M T
MT
Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Luo
S F
SF
Wang
C C
CC
Chien
C S
CS
Chiu
C T
CT
Lin
C C
CC
Yang
C M
CM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005910:Glioma / Q000473:pathology; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D037342:Pertussis Toxin; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D010566:Virulence Factors, Bordetella; D000255:Adenosine Triphosphate; D004247:DNA; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D019311:Staurosporine; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium; D014544:Uridine Triphosphate