Title
Different induction of two plasminogen activator inhibitor 1 mRNA species by phorbol ester in human hepatoma cells
Author
Gaubius Instituut TNO
Bosma, P.J.
Kooistra, T.
Publication year
1991
Abstract
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. Chemicals/CAS: Cycloheximide, 66-81-9; Plasminogen Inactivators; Protein Kinase C, EC 2.7.1.37; RNA, Messenger; Tetradecanoylphorbol Acetate, 16561-29-8
Subject
Blotting, Northern
Carcinoma, Hepatocellular
Cycloheximide
Enzyme Activation
Human
Liver Neoplasms
Plasminogen Inactivators
Protein Kinase C
RNA Processing, Post-Transcriptional
RNA, Messenger
Tetradecanoylphorbol Acetate
Transcription, Genetic
Translation, Genetic
Tumor Cells, Cultured
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TNO identifier
231619
ISSN
0021-9258
Source
Journal of Biological Chemistry, 266 (266), 17845-17849
Document type
article