REGULATION OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR EXPRESSION BY AN ERK1-DEPENDENT SIGNALING PATHWAY IN A SQUAMOUS-CELL CARCINOMA CELL-LINE

The urokinase-type plasminogen activator contributes to tissue remodel ing by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellu lar signal-regulated kinases (ERKs) in the regulation of urokinase-typ e plasminogen activator expression in a squamous cell carcinoma cell l ine (UM-SCC-1) that contains a transcriptionally activated urokinase-t ype plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promo ter, which had progressive 5' deletions or which had been point-mutate d, indicated the requirement of binding sites for AP-1 (-1967) and PEA 3 (-1973) for its maximal activation. Expression of a mutant jun prote in, which lacks the transactivation domain, caused a dose-dependent re pression of a CAT reporter driven by either the urokinase-type plasmin ogen activator promoter or three tandem AP-1 repeats upstream of a thy midine kinase minimal promoter indicating the importance of AP-1-bindi ng transcription factor(s) in the regulation of urokinase-type plasmin ogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotid e spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via pho sphorylation of p62(TCF), but not ERK2, in UM-SCC-1 cells. Moreover, t he expression of a dominant-negative ERK1, but not ERK2, repressed uro kinase-type plasminogen activator promoter activity. Similarly, interf ering with the function of the c-raf serine-threonine kinase, which li es upstream of ERK1, by the expression of a kinase-inactive c-raf repr essed the activity of a CAT reporter driven by either the urokinase-ty pe plasminogen activator promotor or tandem AP-1 repeats. These data s uggest that urokinase-type plasminogen activator expression in UM-SCC- 1 cells is regulated partly by an ERK1, but not ERK2, -dependent signa ling pathway. (C) 1996 Wiley-Liss, Inc.