Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

Abstract

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serumstimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm 2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm 2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G 0 /G 1 to G 2 /M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serumstimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.Extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 1 represent one subfamily of serine/threonine protein kinases collectively referred to as the mitogen-activated protein kinase (MAPK) family. They have the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dual-specificity kinase called MAPK kinase (MAPKK or MKK) or MAPK/ERK kinase (MEK) (reviewed in Refs. 1 and 2). The MEKs upstream of ERKs constitute an evolutionary conserved family of protein kinases that includes at least three highly homologous mammalian isoforms, namely MEK1a, MEK1b, and MEK2 (2). They are highly specific for both of their downstream targets ERK1 and ERK2 (2) and are typically activated by serine/threonine phosphorylation catalyzed by three different classes of upstream kinases: the Raf family of serine/threonine kinases, Raf-1, A-Raf, and B-Raf (3-7), the protooncogene product Mos (8, 9), and the MEK kinase 1 (MEKK1) (10). Despite their high degree of similarity, MEK1a and MEK2...

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