Aims: 

PAI-1 is the major inhibitor of fibrinolysis and elevated plasma PAI-1 levels are associated with greater risk of myocardial infarction (MI). Circulating PAI-1 levels are under genetic control and show circadian variation, with increased levels in the morning, coinciding with the peak in risk of MI. The present study investigated the regulation of mouse and human Pai-1 and the interplay between circadian and hypoxia-induced transcription factors.

Methods: 

Fragments of the mouse and human Pai-1 5'-flanking regions (-707 to +108bp, -796 to +140, resp.) containing canonical E-boxes (CACGTG) and hypoxia-responsive elements (HRE, CACGTA) were cloned into Luciferase reporter vectors. Promoter activity was analysed following over-expression of factors in mouse NIH-3T3 cells.

Results: 

Circadian heterodimeric factors CLOCK and BMAL1 significantly activated (2-fold) the mouse promoter via the E-box at -179 to -174bp. The HRE four bases upstream was activated by hypoxia-induced factor EPAS1 (12-fold), which was significantly augmented by BMAL1 (16-fold). Mutation of this HRE reduced activation by EPAS1 and EPAS1:BMAL1 by ~50%. E-box mutagenesis increased activation of EPAS1 and EPAS1:BMAL1 at the mouse HRE. CLOCK:BMAL1 and EPAS1 also activated the human reporter (3 and 10-fold, resp.) and addition of BMAL1 augmented activation by EPAS1 (30-fold).

Conclusions: 

A conserved mechanism of Pai-1 regulation appears to involve differential binding of circadian and hypoxic factors to the mouse and human proximal promoters. The variation in availability of circadian factors at different times of day may influence the response of Pai-1 expression to hypoxia and contribute to the increased morning incidence of MI. Supported by the BBSRC United Kingdom and the Coulson Trust.