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サイクリンD1発現におけるホスファチジルイノシトール3-キナーゼの役割：多久和典子¹、多久和　陽^1,2　(¹東大・医・細胞分子生理,・²国際科学振興財団)

Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through a Pathway Other than the mTOR-p70S6K Signaling Cascade in Growth Factor-Stimulated NIH3T3 Fibroblasts : Noriko TAKUWA¹ and Yoh TAKUWA² (¹Department of Molecular and Cellular Physiology, University of Tokyo School of Medicine, and ²Foundation for Advancement of International Science (FAIS).)

Phosphatidylinositol (PI) 3-kinase is required for G1 to S phase cell cycle progression stimulated by a variety of growth factors, and is implicated as a regulator for activation of several downstream targets, including p70^S6K. However, molecular mechanisms by which PI 3-kinase is engaged in the activation of the cell cycle machinery is not fully understood. Here we report that a transient expression of wild type p110α catalytic subunit of PI 3-kinase was capable of inducing cyclin D1 protein in quiescent NIH3T3(M17) fibroblasts. This effect of p110 was strongly attenuated by either the PI 3-kinase inhibitor LY294002 or rapamycin, but not by the MEK inhibitor PD98059 or an induced expression of a dominant negative Ras, Ras(N17). The expression of a constitutively active form of Rac, Rac(V12), was also capable of inducing cyclin D1 protein in quiescent NIH3T3(M17) cells. However, Rac was not likely to mediate the effect of wild type p110, since (1) a dominant negative Rac, Rac(N17), failed to inhibit cyclin D1 protein expression when co-expressed with wild type p110, and (2) lamellipodia formation characteristic of Rac(V12) expressing cells was totally absent from the cells overproducing wild type p110. Co-expression with wild type p110 of a dominant negative form of either JNK, c-Akt, PKCε or PKCζ was also ineffective in preventing the induction of cyclin D1 protein. The expression of wild type p110 also greatly potentiated epidermal growth factor (EGF)-stimulated cyclin D1 protein expression. Conversely, the expression of a dominant negative form of either p110 or p85 regulatory subunit of PI 3-kinase strongly inhibited EGF-induced up-regulation of cyclin D1 protein. LY294002 and another PI 3-kinase inhibitor wortmannin completely abrogated EGF-stimulated increases in both mRNA and protein levels of cyclin D1, pRb phosphorylation and S phase entry. However, rapamycin had little inhibitory effect, if any, on either of these events despite potent inhibition of the p70^S6K activity throughout the G1 phase. Similar results were also obtained when the cells were stimulated with other growth stimuli, except that in some of these cases rapamycin was capable of inhibiting DNA synthesis through a mechanism other than inhibition of cyclin D1 expression. These results indicate that PI 3-kinase is both necessary and sufficient for up-regulation of cyclin D1, with the downstream mTOR-p70^S6K signaling pathway differentially required depending on cellular conditions.