| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 87 === Extracellular purine and pyrimidine nucleotides, acting through P2Y receptors, have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the activation of the P2Y receptor on C6 glioma cells responsible for stimulating cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) activation. UTP and ATP produced a similar effect on [3H]thymidine incorporation in a time- and concentration-dependent manner, suggesting the involvement of P2Y2 receptor in mediating the cell proliferation in C6 glioma cells. Furthermore, we examined the mitogenic effects of nucleotides on C6 glioma cells, associated with the activation of MAPK. In response to UTP, both p42 and p44 MAPK isoforms were activated in a time- and concentration-dependent manner using Western blot analysis with an anti-phospho-p42/p44 MAPK antibody. The phosphorylation was transient, reaching maximal levels after 5 min and recovering to basal by 30 min. The mitogenic effect of UTP was mediated via a pertussis toxin-insensitive G protein that involved in activation of MAPK. Both DNA synthesis and phosphorylation of MAPK isoforms in response to UTP were abolished by tyrosine kinase inhibitors, genistein and herbimycin A, as well as protein kinase C (PKC) inhibitors, staurosporine and GF109203X. Removal of Ca2+ by addition of BAPTA/AM and EGTA simultanesimulously significantly inhibited UTP-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation. UTP-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we have also shown that overexpression of dominant negative mutants of Ras (Ras-N17), Raf (Raf301) and MEK1 (MEK-K97R) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of UTP is mediated through a PTX-insensitive G protein-coupled receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP-mediated MAPK activation was modulated by Ca2+, PKC, and tyrosine kinase associated with cell proliferation in cultured C6 glioma cells.
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