Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences.
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 beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, 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 beta-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.
Heaton
J H
JH
Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA. heatonj@umich.edu
Tillmann-Bogush
M
M
Leff
N S
NS
Gelehrter
T D
TD
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000235:genetics*; D005914:Globins / Q000235:genetics; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D010960:Plasminogen Activators / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D015124:8-Bromo Cyclic Adenosine Monophosphate; D005914:Globins; D010960:Plasminogen Activators