Plasminogen activator inhibitor type-1 protein, mRNA and gene transcription are increased by phorbol esters in human rhabdomyosarcoma cells

By use of an enzyme-linked immunosorbent assay, we have found that phorbol 12-myristate 13-acetate (PMA) causes an approximately 10-fold increase in the level of type-1 plasminogen activator inhibitor (PAI-1) accumulated in conditioned medium of the human rhabdomyosarcoma cell line. Half-maximal stimulation occurred at ≃15 nM PMA. The effect was only observed with phorbol esters that are tumor promoting. Maximal levels of secreted PAI-1 were observed 24 h after PMA addition. The increase in secreted PAI-I was preceded by a transient approximately 10-fold increase in intracellular PAI-1 content, maximal at 8 h after PMA addition. There was a 20-fold increase in the cellular level of two 2.3- and 3.4-kilobase PAI-1 mRNAs and a more than 5-fold increase in the PAI-1 gene transcription rate. The protein synthesis inhibitor cycloheximide (10 μg/ml) also increased the level of PAI-1 mRNA, and when both cycloheximide and PMA were used, an additive effect was observed. Cycloheximide changed the ratio between the two PAI-1 mRNAs in favor of the 3.4-kilobase species. Overall, the data show that transcriptional activation of the PAI-1 gene forms part of the pleiotropic responses to tumor-promoting phorbol esters.