Bosma P J, Kooistra T
Gaubius Laboratory TNO, Leiden, The Netherlands.
J Biol Chem. 1991 Sep 25;266(27):17845-9.
In man, the plasminogen activator inhibitor 1 (PAI-1) gene codes for two mRNA species, one of 3.2 kilobases (kb) and the other of 2.4 kb. We report that the protein kinase C activating phorbol ester, phorbol 12-myristate-13-acetate (PMA), causes a different induction of the two PAI-1 mRNA species in the human hepatoma cell line, HepG2. Upon addition of 100 nM PMA, the level of the 3.2-kb PAI-1 mRNA species increased to 25-fold after 3 h, and then declined rapidly. The level of the 2.4-kb species increased more slowly and reached a maximal 18-fold stimulation after 6 h, followed by a gradual decrease towards control levels. Run-on analysis showed that PMA induces a transient 40-fold increase in PAI-1 gene transcription rate. The relative concentration of the two PAI-1 mRNA species in the nuclei of PMA-treated HepG2 cells shifted towards the 2.4-kb form, suggesting that changes in transcription termination site and/or post-transcriptional nuclear processing might contribute to their different accumulation. Also, the two mRNAs differ in turnover rate, with a half-life of about 0.85 h for the 3.2-kb form and a half-life of about 2.5 h for the 2.4-kb form. By itself, cycloheximide had no effect on PAI-1 gene transcription rate or PAI-1 mRNA levels in HepG2. When added 1 h prior to PMA, however, cycloheximide prevented the induction of PAI-1 mRNA, which suggests that PMA exerts its stimulating transcriptional activity through a newly synthesized regulatory protein. When cycloheximide was added 2 h after PMA, when the PAI-1 gene transcription rate was maximally increased, the two PAI-1 mRNAs reached even higher levels than with PMA alone and maximal mRNA levels were maintained for a much longer period (up to 8 h). Thus, ongoing protein synthesis is required for both the induction and the transient nature of the PMA-induced PAI-1 mRNA accumulation. We conclude that the differential accumulation of the two PAI-1 mRNAs by PMA in serum-starved HepG2 cells is due both to changes in transcription termination and/or post-transcriptional nuclear processing and to differences in half-life between the two mRNAs in a process that requires ongoing protein synthesis.
在人类中，纤溶酶原激活物抑制剂1（PAI-1）基因编码两种mRNA，一种为3.2千碱基（kb），另一种为2.4 kb。我们报告，蛋白激酶C激活剂佛波酯，佛波醇12-肉豆蔻酸酯-13-乙酸酯（PMA），在人肝癌细胞系HepG2中对两种PAI-1 mRNA产生不同的诱导作用。加入100 nM PMA后，3.2-kb的PAI-1 mRNA水平在3小时后增加到25倍，然后迅速下降。2.4-kb的mRNA水平增加较慢，在6小时后达到最大18倍的刺激，随后逐渐降至对照水平。核转录分析表明，PMA诱导PAI-1基因转录速率瞬时增加40倍。PMA处理的HepG2细胞核中两种PAI-1 mRNA的相对浓度向2.4-kb形式转变，这表明转录终止位点和/或转录后核加工的变化可能导致它们的不同积累。此外，两种mRNA的周转率不同，3.2-kb形式的半衰期约为0.85小时，2.4-kb形式的半衰期约为2.5小时。单独使用放线菌酮对HepG2细胞中的PAI-1基因转录速率或PAI-1 mRNA水平没有影响。然而，在PMA加入前1小时加入放线菌酮可阻止PAI-1 mRNA的诱导，这表明PMA通过新合成的调节蛋白发挥其刺激转录活性。当在PMA加入2小时后加入放线菌酮时，此时PAI-1基因转录速率已最大程度增加，两种PAI-1 mRNA达到的水平甚至比单独使用PMA时更高，并且最大mRNA水平维持的时间长得多（长达8小时）。因此，持续的蛋白质合成对于PMA诱导的PAI-1 mRNA积累的诱导和瞬时性质都是必需的。我们得出结论，在血清饥饿的HepG2细胞中，PMA导致的两种PAI-1 mRNA的差异积累是由于转录终止和/或转录后核加工的变化以及两种mRNA在需要持续蛋白质合成的过程中半衰期的差异。
