MECHANISM OF GLUCOCORTICOID INDUCTION OF THE RAT PLASMINOGEN-ACTIVATOR INHIBITOR-1 GENE IN HTC RAT HEPATOMA-CELLS - IDENTIFICATION OF CIS-ACTING REGULATORY ELEMENTS

Type 1 plasminogen activator inhibitor (PAI-1) is the major physiologi cal inhibitor of plasminogen activation, inhibiting both tissue- and u rokinase-type plasminogen activators. In HTC rat hepatoma cells, gluco corticoids increase PAI-1 activity, antigen and mRNA accumulation 3- t o 5-fold; this increase is due solely to an increase in the rate of PA I-1 gene transcription. We have identified the cis-acting sequences in the 5'-flanking sequence of the HTC PAI-1 gene that mediate this indu ction. Analysis of a series of hybrid genes containing various portion s of the PAI-1 5'-flanking region fused to the chloramphenicol acetylt ransferase reporter gene transfected into HTC cells localized the regi on involved in the transcriptional regulation by glucocorticoids to be tween -1237 and -764. Electrophoretic mobility shift assays and DNase- I protection assays showed that a glucocorticoid response element (GRE ) 15-mer located at -1212 bound the glucocorticoid receptor DNA-bindin g domain protein in a concentration-dependent manner. Mutations create d within this GRE eliminated its ability both to confer a glucocortico id response and to bind the glucocorticoid receptor. When placed upstr eam of a heterologous promoter in either orientation, this GRE conferr ed glucocorticoid inducibility. We, therefore, conclude that the sole cis-acting sequence required for the glucocorticoid response of the PA I-1 gene in rat HTC hepatoma cells is the GRE at -1212.