Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells.
Regulation of plasminogen activation by plasminogen activator inhibitor type-1 (PAI-1) is a critical feature of many biological processes. Transforming growth factor-beta (TGF-beta) induces PAI-1 mRNA and protein in several types of cultured cells, including Hep G2 cells. The present study was performed to define mechanisms by which PAI-1 gene expression is regulated by TGF-beta. Nuclear run-on assays performed on Hep G2 cells stimulated with TGF-beta for 6 h showed a 3.8-fold increase in PAI-1 gene transcription. TGF-beta increased the half-life of PAI-1 mRNA in Hep G2 cells 2.5-fold over control values. To characterize transcriptional regulatory mechanisms, we constructed chimeric genes containing PAI-1 5'-flanking DNA fused upstream of the bacterial chloramphenicol acetyltransferase gene in the vector pSVOCAT and transfected Hep G2 cells. Promoter deletion analysis demonstrated that sequences between -791 and -328 and -328 to -186 base pairs upstream of the PAI-1 gene cap site contain TGF-beta responsive elements that conferred TGF-beta inducibility in an orientation and position-independent manner. Further characterization of the larger TGF-beta-inducible enhancer (-791 to -328) located a TGF-beta-inducible element at nucleotides -791 to -546 upstream of the PAI-1 gene cap site. These results demonstrate that PAI-1 gene regulation by TGF-beta in Hep G2 cells is mediated both at a transcriptional level by two specific inducible elements, as well as by post-transcriptional mechanisms.
Westerhausen
D R
DR
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Hopkins
W E
WE
Billadello
J J
JJ
D015152:Blotting, Northern; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D015849:Plasminogen Inactivators; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D015291:Transforming Growth Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015849:Plasminogen Inactivators; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D015291:Transforming Growth Factors; D015500:Chloramphenicol O-Acetyltransferase