Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HU-VEC) lambda gtl1 cDNA library. Three overlapping clones were identified by immunologic screening of 10' recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-I mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-i gene is located on human chromosome 7.

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The transforming growth factor-␤ (TGF-␤) family of cytokines and glucocorticoids regulate diverse biological processes through modulating the expression of target genes. Here we report that glucocorticoid receptor (GR) represses TGF-␤ transcriptional activation of the type-1 plasminogen activator inhibitor (PAI-1) gene in a ligand-dependent manner. Similarly, GR represses TGF-␤ activation of the TGF-␤ responsive sequence containing Smad3/4-binding sites. Using mammalian two-hybrid assays, we demonstrate that GR inhibits transcriptional activation by both Smad3 and Smad4 C-terminal activation domains. Finally, we show that GR interacts with Smad3 both in vitro and in vivo. These results suggest a molecular basis for the cross-regulation between glucocorticoid and TGF-␤ signaling pathways.

Incubation of HTC rat hepatoma cells with 8-bromocAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported previously that the 3-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability. By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein.The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs. Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability.Regulation of mRNA stability is an important component of the regulation of gene expression and is known to have a significant role in normal physiology and development (1-5). Our understanding of the regulation of message degradation has been enhanced by the identification of consensus cis-acting sequences that are involved in determining message stability and of some proteins that interact with them (4, 6). Although it is known that many stimuli alter mRNA stability and some cis-acting sequences responsible have been identified, in few cases have trans-acting factors been isolated (2, 7-10). In contrast, a broad spectrum of RNA-binding proteins that are involved in RNA processing, cellular localization, and translation have been identified, and structural domains involved in RNA recognition have been described (11,12). In many cases RNAbinding proteins contain short signature domains that bind RNA and anchor the protein such that functional domains align (13,14). Much less is known about the mechanisms by which RNA-binding proteins regulate mRNA stability.Plasminogen activators (PAs) 1 are serine proteases that catalyze the conversion of plasminogen to the broad spectrum protease, plasmin. Plasmin is the major fibrinolytic en...

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We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at -0.4 U/ml (<4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active a-thrombin and was not obtained with 'y-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.