CONSTITUTIVELY ACTIVE MUTANT OF THE MITOGEN-ACTIVATED PROTEIN-KINASE KINASE MEK1 INDUCES EPITHELIAL DEDIFFERENTIATION AND GROWTH-INHIBITIONIN MADIN-DARBY CANINE KIDNEY-C7 CELLS

Overexpression of a constitutively active mitogen-activated protein ki nase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the p resent study, we used a constitutively active MAPK/extracellular signa l-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the fun ction of the highly conserved MEK1-ERK2 signaling module in renal epit helial cell differentiation and proliferation. Stable expression of co nstitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated 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 abolishe d by the synthetic MEK inhibitor PD098059. Increased ERK2 activation d ue to stable expression of CA-MEK1 in MDCK-C7 cells was associated wit h epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increas ed 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 cel l number/cm(2) after 5 days), This result was confirmed by flow cytome tric 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 comp ared with both untransfected and mock-transfected cells. Taken togethe r, our data demonstrate an association of increased basal and serum-st imulated activity of the MEK1-ERK2 signaling module with epithelial de differentiation and growth inhibition in MDCK-C7 cells. Thus, the MIEK 1-ERK2 signaling pathway could act as a negative regulator of epitheli al differentiation thereby leading to an attenuation of MDCK-C7 cell p roliferation.