Transcriptional regulation of the rat plasminogen activator-inhibitor 1 gene.

Plasminogen activator-inhibitor 1 (PAI-1) is a serine protease inhibitor that inhibits both tissue-type and urokinase-type plasminogen activators. The rat PAI-1 gene is regulated by a number of effectors, including glucocorticoids. To determine the molecular mechanisms involved in the basal and the glucocorticoid-induced expression of the rat PAI-1 gene, the cis-acting sequences and the trans-acting factors involved in the transcription of this gene were analyzed in the HTC rat hepatoma cell line. DNase I protection analyses revealed eight regions within the first 764 base pairs of the rat PAI-1 promoter that interacted specifically with HTC cell nuclear proteins. The proteins that bind to five of the eight footprinted sites were identified as PEA3-, Sp1-, and CTF/NF-1-like proteins using competition electrophoretic mobility shift assays. The expression of fusion genes containing progressive 5$\sp\prime$ deletions of the PAI-1 promoter was analyzed in transient transfection experiments in HTC cells. These studies identified the Sp1 and CTF/NF-1 sites as being important for transcriptional activation. Two of the footprinted sites contain the sequence 5$\sp\prime$-TTTGn (n) TCAAT-3$\sp\prime$, and were shown in competition mobility shift assays to bind the same or related protein(s). Sequences containing these sites were identified in functional studies as transcriptional repressors. The sequence responsible for the glucocorticoid response of the rat PAI-1 gene in HTC cells was shown by others to reside between nucleotides $-$1237 and $-$764. Mobility shift assays and DNase I protection assays demonstrated that the glucocorticoid receptor DNA-binding domain (GR-DBD) binds to a glucocorticoid response element (GRE) located within these sequences. Mutations of the GRE were made and tested in functional studies by others. Mobility shift assays identified that the mutated GRE sequences that retained the ability to induce transcription in response to glucocorticoids were able to bind the GR-DBD. The mutated GRE sequences that failed to induce transcription in response to glucocorticoids failed to bind the GR-DBD. These data confirm that this GRE sequence is responsible for the glucocorticoid response of the rat PAI-1 gene in HTC cells, and strongly suggest that the glucocorticoid receptor mediates the response.PhDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105719/1/9208570.pdfDescription of 9208570.pdf : Restricted to UM users only