CYCLIC-NUCLEOTIDE REGULATION OF TYPE-1 PLASMINOGEN ACTIVATOR-INHIBITOR MESSENGER-RNA STABILITY IN RAT HEPATOMA-CELLS - IDENTIFICATION OF CIS-ACTING SEQUENCES

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma ce lls with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramat ic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA, Although 8 bromo-cAMP causes a modest d ecrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequ ences from the rat PAI-1 cDNA and the mouse beta-globin gene and exami ned the effect of cyclic nucleotides on the decay rate of these transc ripts. The mRNA transcribed from the beta-globin gene is stable and no t cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslate d region (UTR) is destabilized in the presence of 8-bromo-cAMP, sugges ting that this response is mediated by sequences in the PAI-1 3'-UTR., Analyses by deletion of sequences from this chimeric construct indica te that, whereas more than one region of the PAI-1 3'-UTR can confer c yclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence a lone is sufficient to do so. Insertion of PAI-1 sequences within the b eta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 m RNA can confer cyclic nucleotide regulation of stability on a heterolo gous transcript, suggesting that this sequence may play a major role i n hormonal regulation of PAI-1 mRNA stability.