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Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease 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 sequences from the rat PAI-1 cDNA and the mouse ␤-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the ␤-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the ␤-globin coding region and the PAI-1 3-untranslated region (UTR) is destabilized in the presence of 8-bromocAMP, suggesting that this response is mediated by sequences in the PAI-1 3-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3-UTR can confer cyclic nucleotide responsiveness, the 3-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the ␤-globin 3-UTR confirms that the 3-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.Eukaryotic gene expression is determined not only by the rate of gene transcription, but also by the rate at which the transcript is degraded. The steady state concentration of a particular mRNA, as well as the rate at which a new transcriptionally induced steady state is attained, is directly related to the message half-life (1, 2). Numerous recent studies have been aimed at understanding the role of specific sequences within the mRNA in determining basal transcript stability and have led to the identification of consensus sequences that confer stability or instability to a transcript (2, 3). Although it is known that many stimuli alter mRNA stability, little is known about either the cis-acting sequences in the RNA or the cellular factors involved in regulation of message decay.Plasminogen activators (PAs) 1 are serine proteases that are critical for thrombolysis and also play a key role in other physiological functions involving tissue remodeling. Plasminogen activator-inhibitors (PAIs) are specific inhibitors of PAs and are members of the serine protease inhibitor (serpin) family of proteins. Type-1 PAI (PAI-1) is a 50 kDa glycoprotein found in plasma, platelets, and a variety of cell types; its expression is regulated by growth factors, cytokines, and hormones, including agents that alter cellular cAMP levels (4).HTC rat hepatoma cells synthesize and secrete tissue-type plasminogen activator (tPA) and P...

Plasminogen activators (PAs) 1 are serine proteases that catalyze the conversion of the zymogen plasminogen to plasmin, a broad spectrum endopeptidase that is responsible for intravascular fibrinolysis (1). This protein is also known to play a major role in biological processes involving localized proteolysis of extracellular matrix, such as tissue remodeling and tumor cell invasion and metastasis (2). Type-1 plasminogen activatorinhibitor (PAI-1), a 50-kDa glycoprotein, is a major regulator of plasminogen activation (3, 4). PAI-1 is synthesized in a variety of cell types and its expression is regulated by growth factors and hormones, including agents that elevate intracellular cAMP levels (5-8).In HTC rat hepatoma cells, the cyclic nucleotide analogue 8-bromo-cAMP, together with the phosphodiesterase inhibitor isobutylmethylxanthine (designated cA), increases tissue type PA activity more than 50-fold primarily by decreasing PAI-1 mRNA by 90% and protein by 60 -70%. The decrease in PAI-1 mRNA is due to a 60% decrease in the rate of PAI-1 gene transcription and, more importantly, a 3-fold increase in the rate of PAI-1 mRNA decay (8, 9). Utilizing HTC cells stably transfected with chimeric constructs containing portions of the mouse ␤-globin gene and rat PAI-1 cDNA, the 1730-nucleotide (nt) PAI-1 3Ј-untranslated region (UTR) (nt 1331-3060) was shown to contain sequences that mediate the cA-induced destabilization (10). Furthermore, results obtained from deletion and insertion analyses using a series of ␤-globin coding region: PAI-1 3Ј-UTR chimeric constructs demonstrated that at least two regions within the PAI-1 3Ј-UTR mediate the cA effect. One of these regions is the 3Ј-most 134 nt, from position 2926 to 3060, of the PAI-1 mRNA. This 134-nt sequence includes a 75-nt U-rich region present at its 5Ј end and a 24-nt A-rich region at its 3Ј end (Fig. 1A) (10, 11).The decay rates of many mRNAs have been shown to be regulated by a variety of external stimuli including hormones, growth factors, and agents that elevate intracellular cAMP levels (see Refs. 12 and 13 for review). Studies aimed at elucidating the mechanism(s) involved in regulating mRNA stability have identified a number of potential cis-acting sequences and/or trans-acting factors (12, 13); however, the molecular basis for the cyclic nucleotides regulation of mRNA stability remains largely undefined.The aim of the present study was to further elucidate the mechanism(s) involved in the cyclic nucleotide-induced destabilization of PAI-1 mRNA in rat HTC cells. To this end, studies were conducted to identify the cytosolic factors that bind to the 3Ј-most 134 nt of the PAI-1 mRNA, to characterize the specificity and binding sites for these factors, and to determine their role in cA-induced PAI-1 mRNA destabilization. Results from ultraviolet (UV) cross-linking analyses demonstrate that specific RNA-protein complexes of ϳ38, 50, 53, 61, 65, and 76 kDa form with the 134-nt sequence, while RNA electrophoretic mobility shift analyses (R-EMSAs) demonstrate the f...

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