Stimulation of urokinase expression by TNF-α requires the activation of binding sites for the AP-1 and PEA3 transcription factors

The urokinase-type plasminogen activator plays a central role in tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. Urokinase expression is transcriptionally regulated by a variety of cytokines including TNF-α. The present study was undertaken to identify key transcription factor binding sites in the urokinase promoter necessary for the TNF-α-dependent induction of urokinase expression. TNF-α treatment of a squamous cell carcinoma cell line, UM-SCC-1, which produces no detectable TNF-α, led to a dose-dependent increase in urokinase secretion, thus reflecting a more abundant mRNA. Transient transfections of UM-SCC-1 cells with a CAT reporter driven by 5′ deletion fragments of the urokinase promoter indicated that a sequence spanning -2109 to -1870, which contained binding sites for AP-1 and PEA3 was required for the stimulation by TNF-α. Mutation of an AP-1 binding site at -1967 and a PEA3 motif at -1973 completely abrogated the inductive effect of TNF-α on urokinase promoter activity. Mobility shift assays indicated the presence of a jun-containing factor(s) which bound specifically to the AP-1 sequence present in the urokinase promoter. The amount and/or activity of this factor(s) was greatly enhanced by TNF-α treatment. UM-SCC-1 cells transiently transfected with a CAT reporter driven by 3 tandem AP-1 binding sites demonstrated increased CAT activity following TNF-α treatment. Thus, the induction of urokinase expression by TNF-α is likely to involve the altered expression and/or activity of transcription factors which bind to the AP-1 and PEA3 target sequences in the urokinase promoter.