Tumor Necrosis Factor α Activates the Human Plasminogen Activator Inhibitor-1 Gene through a Distal Nuclear Factor κB Site

Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of plasminogen activation and likely plays important roles in coronary thrombosis and arteriosclerosis. Tumor necrosis factor-α (TNFα) is one of many recognized physiological regulators of PAI-1 expression and may contribute to elevated plasma PAI-1 levels in sepsis and obesity. Although TNFα is a potent inducer of PAI-1 expression in vitro and in vivo, the precise location of the TNFα response site in the PAI-1 promoter has yet to be determined. Transient transfection studies using luciferase reporter constructs containing PAI-1 promoter sequence up to 6.4 kb failed to detect a response to TNFα. Moreover, TNFα failed to induce expression of enhanced green fluorescent protein under the control of a 2.9-kb human PAI-1 promoter in transgenic mice, although endogenous murine PAI-1 was strongly induced. These data suggested that the TNFα response element in the PAI-1 gene is remote from the proximal promoter region. In this study, seven candidate regulatory regions were identified using cross-species sequence homology analysis as well as DNase I-hypersensitive site analysis. We identified a 5′ distal TNFα-responsive enhancer of the PAI-1 gene located 15 kb upstream of the transcription start site containing a conserved NFκB-binding site that mediates the response to TNFα. This newly recognized site is fully capable of binding NFκB subunits p50 and p65, whereas overexpression of the NFκB inhibitor IκB prevents TNFα-induced activation of this enhancer element.