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 formation at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitro invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-activated protein kinases (MAPKs) regulate u-PAR expression in two cultured colon cancer cell lines. An in-gel kinase assay showed that ERK1 activity was considerably higher in RKO cells, which display > or = 10(5) receptors/cell, than the GEO cells which have approximately 10(4) urokinase receptors per cell. The expression of either an ERK-inactivating phosphatase (CL100), or a kinase-defective ERK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constitutive ERK1 activity in RKO cells was largely a result of an activated MEK1. Further, treatment of RKO cells with a specific inhibitor (PD 098059) of MEK1 activation, which diminished ERK1 activity, reduced the amount of urokinase specifically bound to the cell surface and this was associated with reduced laminin degradation. The expression of a dominant negative c-Raf-1 also reduced u-PAR promoter activity suggesting that MEK1 activation involved an activator at, or upstream, of this serine-threonine kinase. Transfection of the u-PAR-deficient GEO cells with a constitutively activated MEK1 expression construct up-regulated u-PAR promoter activity. Similarly treatment of GEO cells with a phosphatase inhibitor (sodium vanadate) caused a dose-dependent increase in ERK1 activity which paralleled increased cell surface binding of urokinase. 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.
Lengyel
E
E
Department of Tumor Biology/Head and Neck Surgery, MD Anderson Cancer Center, Houston, Texas, USA.
Wang
H
H
Gum
R
R
Simon
C
C
Wang
Y
Y
Boyd
D
D
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins; D003110:Colonic Neoplasms / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D011905:Genes, ras / Q000235:genetics; D017874:Immediate-Early Proteins; D007797:Laminin / Q000187:drug effects / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D010960:Plasminogen Activators / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D017874:Immediate-Early Proteins; D007797:Laminin; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D010960:Plasminogen Activators; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one