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

Herbert Schramek,Elisabeth Feifel,Edel Healy,Verena Pollack

doi:10.1074/jbc.272.17.11426

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 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 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/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 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 G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated 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.

Highlights

A mitogen-activated protein kinase (MAPK) kinase (MKK or MAPK/ERK kinase (MEK)) recently cloned from Chinese hamster lung fibroblasts [13] encodes a protein of 45 kDa that exhibits a high identity with the Xenopus [14] and murine [15] amino acid sequences
We report that stable expression of a hemagglutinin epitope-tagged constitutively active MEK1 mutant (S218D/S222D mutant) in MDCK-C7 cells leads to epithelial dedifferentiation as well as to a reduction of cell proliferation
With respect to the activation of different members of the MAPK family of protein kinases, we have recently reported that these dedifferentiated MDCKC7Focus (MDCK-C7F) cells show increased basal and serumstimulated ERK2 stimulation but slightly decreased basal and anisomycin-induced Jun NH2-terminal kinase 1 (JNK1) activity [22]

Summary

Introduction

A MAPK kinase (MKK or MEK) recently cloned from Chinese hamster lung fibroblasts [13] encodes a protein of 45 kDa that exhibits a high identity with the Xenopus [14] and murine [15] amino acid sequences. Stable Transfection of MDCK-C7 Cells with CA-MEK1 Is Associated with Increased ERK1/2 Phosphorylation and Increased ERK2 Activity—When grown for 2 weeks in alkaline (pH 7.7) culture medium, cloned MDCK-C7 cells dedifferentiate, exhibit spindle-shaped morphology, and lack contact inhibition as well as monolayer formation [22, 23].

Results

Conclusion