Tomono M, Toyoshima K, Ito M, Amano H
Department of Biochemistry, Sakura Hospital, Toho University School of Medicine, Tokyo, Japan.
Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):675-80. doi: 10.1042/bj3170675.
DNA synthesis was measured 16 h after stimulation of Swiss 3T3 fibroblasts in the resting phase with various growth factors (platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid and thrombin). When extracellular Ca2+ was chelated by EGTA, or when the influx of Ca2+ from outside to inside the cell was blocked by cobalt, DNA synthesis was completely inhibited. As there was no effect whatsoever on DNA synthesis when Ca2+ was chelated, or when the influx of Ca2+ was blocked up to the first 4 h after growth stimulation, it was concluded that, at an early stage, Ca2+ influx from outside to inside the cell is not related to the transition from the G1 to the S phase. A Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) had no effect on DNA synthesis. However, cyclosporin A and FK-506, which are inhibitors of Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin), markedly inhibited DNA synthesis stimulated by all of the growth factors. These results indicate that calcineurin plays a role, not only in activation of T-cells of the immune system in the initial phase, but also in DNA synthesis in fibroblasts. It was concluded that Ca2+ influx from outside to inside the cell during the mid-to-late G1 phase, followed by calcineurin activation, is essential as a mechanism of growth signal transduction.
在静止期用各种生长因子（血小板衍生生长因子、成纤维细胞生长因子、溶血磷脂酸和凝血酶）刺激瑞士3T3成纤维细胞16小时后，测量DNA合成。当细胞外Ca2+被乙二醇双四乙酸（EGTA）螯合时，或者当细胞外Ca2+流入细胞内的过程被钴阻断时，DNA合成被完全抑制。由于在生长因子刺激后的最初4小时内，螯合Ca2+或阻断Ca2+流入对DNA合成没有任何影响，因此得出结论，在早期阶段，细胞外Ca2+流入细胞内与从G1期向S期的转变无关。一种Ca2+/钙调蛋白依赖性蛋白激酶II抑制剂（KN-62）对DNA合成没有影响。然而，作为Ca2+/钙调蛋白依赖性蛋白磷酸酶2B（钙调神经磷酸酶）抑制剂的环孢素A和FK-506，显著抑制了所有生长因子刺激的DNA合成。这些结果表明，钙调神经磷酸酶不仅在初始阶段免疫系统T细胞的激活中发挥作用，而且在成纤维细胞的DNA合成中也发挥作用。得出的结论是，在G1期中期到后期，细胞外Ca2+流入细胞内，随后钙调神经磷酸酶被激活，作为生长信号转导机制是必不可少的。
