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 physiological inhibitor of plasminogen activation, inhibiting both tissue- and urokinase-type plasminogen activators. In HTC rat hepatoma cells, glucocorticoids increase PAI-1 activity, antigen and mRNA accumulation 3- to 5-fold; this increase is due solely to an increase in the rate of PAI-1 gene transcription. We have identified the cis-acting sequences in the 5'-flanking sequence of the HTC PAI-1 gene that mediate this induction. Analysis of a series of hybrid genes containing various portions of the PAI-1 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene transfected into HTC cells localized the region involved in the transcriptional regulation by glucocorticoids to between -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-binding domain protein in a concentration-dependent manner. Mutations created within this GRE eliminated its ability both to confer a glucocorticoid response and to bind the glucocorticoid receptor. When placed upstream of a heterologous promoter in either orientation, this GRE conferred glucocorticoid inducibility. We, therefore, conclude that the sole cis-acting sequence required for the glucocorticoid response of the PAI-1 gene in rat HTC hepatoma cells is the GRE at -1212.
Bruzdzinski
C J
CJ
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618.
Johnson
M R
MR
Goble
C A
CA
Winograd
S S
SS
Gelehrter
T D
TD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase