DIFFERENT REGULATION OF PLASMINOGEN-ACTIVATOR INHIBITOR-2 GENE-EXPRESSION BY PHORBOL ESTER AND CAMP IN HUMAN MYELOID-LEUKEMIA CELL-LINE PL-21

Previous studies have shown that protein kinase C (PKC) activators and dibutyryl cyclic AMP (Bt2cAMP) synergistically increase the antigen l evel of plasminogen activator inhibitor type-2 (PAI-2) in a human myel oid leukemia cell line PL-21. To clarify the mechanism, PAI-2 gene exp ression induced by phorbol myristate acetate (PMA), a PKC activator, a nd Bt2cAMP was investigated by Northern blot hybridization using a PAI -2 cDNA probe cloned from a human placental library. The level of PAI- 2 mRNA was markedly increased in response to PMA and reached a maximum 5-9 h after stimulation. Nuclear run-on assay revealed an increase in PAI-2 gene transcription in PMA-treated cells. The induction was inhi bited by inhibiting de novo protein synthesis with cycloheximide (CHX) . cAMP also increased PAI-2 mRNA level in a dose-dependent manner. The increase began within 2 hours and, contrary to the case of PMA, the m RNA levels were maintained. Moreover, cAMP-induced increase in PAI-2 m RNA was not inhibited by CHX, rather enhanced. PMA and cAMP synergisti cally induced PAI-2 gene expression, which was completely inhibited by CHX. The cells pretreated with PMA for 24 h did not any more respond to stimulation with PMA but responded to cAMP and PAI-2 mRNA level was increased. The apparent half-life of constitutive level PAI-2 mRNA in PL-21 cells, determined by actinomycin-D-decay experiments, was appro ximately 2 h. Those induced by PMA and cAMP were approximately 5 h and 2 h, respectively. These data suggest that PAI-2 mRNA induced by PMA is relatively stable and the expression requires de novo protein synth esis, whereas cAMP increases PAI-2 mRNA level without affecting the st ability and the induction does not require de novo protein synthesis. Judging from these data, PAI-2 gene expression appears to be different ly regulated by the PKC and cAMP signalling pathways.