Stimulation of urokinase expression by TNF-alpha 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-alpha. The present study was undertaken to identify key transcription factor binding sites in the urokinase promoter necessary for the TNF-alpha-dependent induction of urokinase expression. TNF-alpha treatment of a squamous cell carcinoma cell line, UM-SCC-1, which produces no detectable TNF-alpha, 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-alpha. Mutation of an AP-1 binding site at -1967 and a PEA3 motif at -1973 completely abrogated the inductive effect of TNF-alpha 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-alpha 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-alpha treatment. Thus, the induction of urokinase expression by TNF-alpha 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.
Lengyel
E
E
Department of Tumor Biology, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Klostergaard
J
J
Boyd
D
D
D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C065788:transcription factor PEA3; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D014568:Urokinase-Type Plasminogen Activator