Trejo J, Chambard J C, Karin M, Brown J H
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Mol Cell Biol. 1992 Oct;12(10):4742-50. doi: 10.1128/mcb.12.10.4742-4750.1992.
Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.
毒蕈碱型胆碱能受体或凝血酶受体的激活会增加磷酸肌醇代谢、钙离子动员以及蛋白激酶C的重新分布，并在1321N1细胞中诱导c-fos mRNA和c-jun mRNA迅速短暂增加。为了确定卡巴胆碱和凝血酶诱导的c-fos和c-jun mRNA增加是否足以刺激AP-1介导的反式激活，将携带两个十四烷酰佛波醇乙酸酯反应元件拷贝和萤火虫荧光素酶基因的报告基因转染到1321N1细胞中。凝血酶在刺激AP-1介导的反式激活方面比卡巴胆碱显著更有效。为了确定卡巴胆碱和凝血酶诱导的AP-1活性差异背后的因素，研究了编码AP-1组分的fos和jun家族成员。卡巴胆碱和凝血酶在急性和24小时时间进程中对c-fos、fosB、fra-2、junB和junD的表达具有相似的影响。然而，卡巴胆碱仅导致c-jun的短暂诱导（在0.5小时达到最大值），而凝血酶诱导c-jun mRNA的双相增加——在0.5小时出现初始峰值，在12小时出现第二个更持久的增加。凝血酶而非卡巴胆碱还诱导fra-1 mRNA的后期增加，在12小时达到峰值。c-jun mRNA的二次增加与c-Jun蛋白水平和AP-1 DNA结合活性的显著增加相关。在凝血酶作用30分钟后加入拮抗剂水蛭素可阻止c-jun和fra-1 mRNA的后期诱导，这导致凝血酶刺激的c-Jun蛋白、AP-1 DNA结合活性和AP-1介导的反式激活增加消失。这些发现表明，c-fos和c-jun mRNA的快速短暂传导不足以诱导基因转录的显著变化，而c-jun mRNA的持续增加以及可能fra-1 mRNA的后期诱导是产生AP-1 DNA结合活性和通过AP-1进行反式激活所必需的。
