Regulatory elements in the plasminogen activator inhibitor type 2 promoter

Abstract

This study examined the regulation of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in response to the cytokine tumour necrosis factor-? (TNF), and also involved the identification of promoter elements involved in regulation of PAI-2 transcription. Initially, DNase I hypersensitivity analyses were used to probe a 9.6 kb region of the PAI-2 gene in untreated and TNF treated U937 and MM6 macrophage cells, to look for TNF-specific regulatory regions. However experiments revealed no DNase I hypersensitive sites exclusively identified in the PAI-2 promoter from TNF treated cells. Therefore an alternative approach to study TNF regulation of the PAI-2 gene was undertaken. It has been established for a number of genes that TNF induction occurs via activation and binding of NF-kB to promoter consensus recognition elements. The potential involvement of two NF-KB-like sites at positions -400 and -270 in the response of the PAI-2 gene to TNF was investigated by reporter gene assays using PAI-2 deletion promoter constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Constructs containing mutations in the -400 and -270 NF-KB-like sites were also examined. In both U937 macrophage and HT1080 fibrosarcoma cells, TNF had no effect on PAI-2 directed CAT reporter gene activity of these promoter constructs. There by indicating that a TNF response element was not present in the 990 bp of the promoter region examined. Mutation of the -270 NF-KB-like site had no affect on the level of CAT reporter gene activity. In contrast, mutation of the -400 site resulted in loss of CAT activity, suggesting that it was essential for basal PAI-2 transcription. This element was designated the PAI-2 -400 transcriptional regulatory motif (TRM). Electrophoretic mobility shift assays (EMSAs) demonstrated three PAI-2 -400 TRM DNA binding proteins (P1, P2 and P3) in U937, HT1080 and HeLa cellular protein extracts. Competitive EMSA and supershift experiments confirmed that none of these DNA binding proteins was NF-kB. The genes encoding these DNA binding proteins were targeted by screening of a cDNA library with the -400 TRM oligonucleotide probe. Expression screening resulted in the isolation of four clones. Three of the clones (1ai, 1ci and 6ii) encoded polypeptides which were specific for the PAI-2 -400 TRM by competitive EMSA analyses.