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Nucleic Acids Research, 1991, Vol. 19, No. 14 3881-3886
© 1991

MOLECULAR BIOLOGY

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter

Elisabeth Cousin, Robert L. Medcalf, Gabriela E. Bergonzelli and Egbert K.O. Kruithof

Central Hematology Laboratory, Medical University School CHUV, 1011 Lausanne, Switzerland

Received April 29, 1991. Revised June 26, 1991. Accepted June 26, 1991.

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly Induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-2 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase In CAT expression from all constructs except from the construct shortened to position -91. DNAsei protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction.


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