ELEVATED UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTOR EXPRESSION IN A COLON-CANCER CELL-LINE IS DUE TO A CONSTITUTIVELY ACTIVATED EXTRACELLULAR SIGNAL-REGULATED KINASE-1-DEPENDENT SIGNALING CASCADE

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix degradation in part by accelerating plasmin forma tion at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitr o invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regu lated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-acti vated protein kinases (MAPKs) regulate u-PAR expression in two culture d colon cancer cell lines, An in-gel kinase assay showed that ERK1 act ivity was considerably higher in RKO cells, which display greater than or equal to 10(4) receptors/cell, than the GEO cells which have simil ar or equal to 10(4) urokinase receptors per cell. The expression of e ither an ERK-inactivating phosphatase (CL100), or a kinase-defective E RK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constituti ve ERK1 activity in RKO cells was largely a result of an activated MEK 1. Further, treatment of RKO cells with a specific inhibitor (PD 09805 9) of MEK1 activation, which diminished ERK1 activity, reduced the amo unt of urokinase specifically bound to the cell surface and this was a ssociated with reduced laminin degradation. The expression of a domina nt negative c-Raf-l also reduced u-PAR promoter activity suggesting th at MEK1 activation involved an activator at, or upstream, of this seri ne-threonine kinase, Transfection of the u-PAR-deficient GEO cells wit h a constitutively activated MEK1 expression construct upregulated u-P AR promoter activity. Similarly treatment of GEO cells,vith a phosphat ase inhibitor (sodium vanadate) caused a dose-dependent increase in ER K1 activity which paralleled increased cell surface binding of urokina se. Taken together, these data suggest that elevated u-PAR expression, in at least a sub-population of colon cancer, is partly a consequence of a constitutively activated ERK-1-dependent signaling cascade.