Tomono M, Toyoshima K, Ito M, Amano H, Kiss Z
Department of Biochemistry, Sakura Hospital, Toho University School of Medicine, Tokyo, Japan.
Arch Biochem Biophys. 1998 May 15;353(2):374-8. doi: 10.1006/abbi.1998.0667.
In Swiss 3T3 fibroblasts, growth factor-stimulated progression from G1 to S phase involves activation of the Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (calcineurin). Here we report that both cobalt and the calcium chelator EGTA, inhibitors of calcium uptake, as well as cyclosporin A and FK-506, specific inhibitors of calcineurin function, abolished fibroblast growth factor (FGF)-induced expression of cyclins A and E, but not cyclin D1. At 0.1 microM concentration cyclosporin A completely blocked FGF-induced expression of cyclins E and A and it inhibited FGF-stimulated DNA synthesis by 40%; full inhibition of DNA synthesis required 10 microM cyclosporin A. PD 98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase, and hemicholinium-3, an inhibitor of FGF-induced MAP kinase activity, did not inhibit the stimulatory effect of FGF on the expression of cyclin E. On the other hand, the inhibitory effect of 0.1 microM cyclosporin A on FGF-stimulated DNA synthesis was additive with that of hemicholinium-3, suggesting that the two inhibitors acted by different mechanisms. The inhibitors of calcineurin and calcium uptake also completely blocked the stimulatory effects of lysophosphatidic acid on the expression of cyclins E and A, but not cyclin D1. The results suggest that FGF- or lysophosphatidic acid-induced transcription of cyclin A and cyclin E genes is mediated by calcineurin involving a MAP kinase-independent mechanism and that increased expression of cyclins A and E is required for the maximal stimulatory effects of these mitogens on DNA synthesis.
在瑞士3T3成纤维细胞中，生长因子刺激的从G1期到S期的进程涉及钙调神经磷酸酶（钙神经素）的激活，钙调神经磷酸酶是一种依赖Ca2+/钙调蛋白的丝氨酸/苏氨酸特异性蛋白磷酸酶2B。在此我们报告，钙摄取抑制剂钴和钙螯合剂EGTA，以及钙调神经磷酸酶功能的特异性抑制剂环孢菌素A和FK-506，均可消除成纤维细胞生长因子（FGF）诱导的细胞周期蛋白A和E的表达，但不影响细胞周期蛋白D1的表达。在0.1微摩尔浓度下，环孢菌素A完全阻断FGF诱导的细胞周期蛋白E和A的表达，并抑制FGF刺激的DNA合成达40%；DNA合成的完全抑制需要10微摩尔的环孢菌素A。丝裂原活化蛋白（MAP）激酶激酶抑制剂PD 98059和FGF诱导的MAP激酶活性抑制剂半胱氨酸-3，均不抑制FGF对细胞周期蛋白E表达的刺激作用。另一方面，0.1微摩尔环孢菌素A对FGF刺激的DNA合成的抑制作用与半胱氨酸-3的抑制作用具有加和性，这表明这两种抑制剂的作用机制不同。钙调神经磷酸酶和钙摄取抑制剂也完全阻断了溶血磷脂酸对细胞周期蛋白E和A表达的刺激作用，但不影响细胞周期蛋白D1的表达。结果表明，FGF或溶血磷脂酸诱导的细胞周期蛋白A和细胞周期蛋白E基因的转录是由钙调神经磷酸酶介导的，涉及一种不依赖MAP激酶的机制，并且细胞周期蛋白A和E表达的增加是这些有丝分裂原对DNA合成产生最大刺激作用所必需的。
