Plasminogen activator inhibitor type 2 (PAI-2) is a member of a large family of structurally similar yet functionally diverse proteins called serine protease inhibitors or serpins. Extracellular PAI-2 inhibits urokinase, a serine protease that degrades the extracellular matrix, while intracellular PAI-2 has a cytoprotective action. In vivo, PAI-2 is expressed selectively by only a few cell types and is induced by a variety of inflammatory mediators and other factors, indicating the existence of precise mechanisms to regulate both the tissue-specific and inducible expression of this gene. Previous studies have demonstrated that transcription from the human PAI-2 gene promoter is controlled by three regulatory domains - a proximal promoter, an upstream silencer and a distal transactivator. While transcription from the PAI-2 promoter in PAI-2 non-expressing cells is specifically repressed by the silencer element, the transactivator region serves to de-repress transcription from the PAI-2 promoter in PAI-2 expressing cells.
In the present study, the cis-acting elements and trans-acting factors mediating transactivator function have been investigated. It is shown that a distal AP-1 site is both necessary and sufficient for PAI-2 transactivator function. Furthermore it is demonstrated that this site specifically binds the Fos transcription factor family member FosB in PAI-2 expressing U937 cells but not PAI-2 non-expressing HeLa cells, and that over-expression of FosB (or the Jun transcription factor family member c-Jun) in HeLa cells is sufficient to cause de-repression of transcription from the PAI-2 promoter in these cells. Finally, preliminary evidence is presented that over-expression of FosB or c-Jun is sufficient to induce PAI-2 mRNA in HeLa cells.
Expression of the murine PAI-2 gene, like its human counterpart, is also regulated at the level of transcription, yet no studies to date have examined the molecular basis for the transcriptional regulation of the murine PAI-2 gene. To address this shortcoming 4.48 kb of the murine PAI-2 promoter was sequenced and characterised by reporter gene analysis in the murine RAW 264.7 macrophage cell line. Analysis of the murine PAI-2 promoter sequence identified several potential cw-acting regulatory elements. Comparison of the nucleotide sequences of the murine and human PAI-2 promoters revealed five regions of extensive sequence similarity between the two promoters, with several key regulatory cw-acting elements in the human PAI-2 promoter also identified in the mouse.
Reporter gene analysis showed that the murine PAI-2 promoter, like the human PAI-2 promoter, contained a PMA- (and LPS-) inducible proximal promoter region and an upstream silencer region. Mutation and deletion studies targeting the murine equivalent of the human PAI-2 promoter silencer element showed that this element is not responsible for silencing the murine PAI-2 promoter. Reporter gene analysis also revealed that a potent LPS responsive element was present in a region of the murine PAI-2 promoter not previously implicated in the transcriptional regulation of the human PAI-2 gene. To characterise this element, mutations targeting several candidate cis-acting regulatory elements in a region of the murine PAI-2 promoter between nucleotides -539 and -189 were examined for their effect on reporter gene activity in murine RAW 264.7 macrophage cells. These studies indicated that a C/EBP site at -203/-192 was essential for LPS-inducible transcription from the murine PAI-2 promoter. Gel shift analysis showed that this region was specifically bound by an LPS-inducible factor in RAW 264.7 nuclear extracts, identified by gel supershift assay to be C/EBPβ. Transient transfection of RAW 264.7 cells with a PAI-2 promoter-reporter gene construct deleted to nucleotide position -212, however, only partially restored LPS-inducibility, indicating that one or more additional cis-acting regulatory elements mediating the maximum response of the murine PAI-2 promoter to LPS was located between nucleotide -539 and the -203/-192 C/EBPβ site.
In summary, these findings have identified new cis-acting regulatory elements and trans-acting factors mediating transcription from the human and murine PAI-2 gene promoters. Together they make an important contribution to the development of an evolving model describing the tissue-specific and inducible regulation of transcription of the PAI-2 gene.