Cloning of the 5′ regulatory regions and functional characterization of the core promoters of ovine PLAU (u-PA) and SERPIN1 (PAI-1)

Abstract

The activation of plasminogen plays a crucial role in various extracellular proteolytic events (fibrinolysis, cell migration, ovulation and involution of the mammary gland). In the present study we describe the isolation of the 5′ proximal and distal promoter regions of ovine PLAU (urokinase plasminogen activator, u-PA) and SERPIN1 (plasminogen activator inhibitor 1, PAI-1) genes for the first time in ruminants. Analysis of the 5.645 kb 5′-flanking region of u-PA revealed a putative TATA-less promoter. In contrast the isolated 2.787 kb 5′-flanking region of PAI-1 included a TATA-box. It should be noted that both genes lack the initiator motif around the transcription start site. The two genes share a number of transcription factor binding sites, namely Nuclear Factor-kappa B, Stimulating Protein 1 and Activating protein 1, suggesting co-expression of the two genes. Moreover, additional, not shared, transcription factor binding sites were identified in u-PA and PAI-1. More important of these are the cis-regulatory elements for plasminogen activator inhibitor 2 located in the distal promoter region of u-PA, suggesting an involvement of the other specific inhibitor in the regulation of ovine u-PA gene expression, and the three stress response elements sites present in the proximal and distal promoter of PAI-1. Different genomic fragments of the two 5′ flanking regions were directionally cloned into a suitable reporter vector upstream of a promoter-less luciferase gene. Transient transfection into bovine mammary epithelial (BME-UV) cells demonstrated that the regions of − 384/+27 and − 382/+22 for the u-PA and PAI-1genes, respectively, potentially function as core promoter regions.