Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells

Abstract

Schramek, Herbert, Elisabeth Feifel, Ingrid Marschitz, Nadejda Golochtchapova, Gerhard Gstraunthaler, and Roberto Montesano. Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells. Am J Physiol Cell Physiol 285: C652-C661, 2003; 10.1152 10. /ajpcell.00463.2002tive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cystlike structures in collagen gels and induces an invasive, myofibroblastlike phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, ␤-catenin, and cytokeratin, by the loss of ␣-smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelialmesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cystlike epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures.

The microenvironmental determinants for kidney epithelial cyst morphogenesis

“…Because of the simplicity of its morphogenetic process, MDCK cyst formation is an ideal model for determining the cellular events critical for epithelial morphogenesis and the molecular mechanism(s) governing it. The signaling involved in MDCK cyst formation has emerged from various studies (Mangoo-Karim et al, 1989;Orellana and Marfella-Scivittaro, 2000;O'Brien et al, 2001;Schramek et al, 2003;Yu et al, 2005). The importance of the microenvironment in cystogenesis is well documented (Lin and Bissell, 1993;Kleinman et al, 2003).…”

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells

“…18,41 Especially in the renal tubular system, strong evidence exists for an important function of the MEK1/2-ERK1/2 signaling module in EMT. 10,[42][43][44] Furthermore, it has been reported that OSM represents a strong stimulator of both ERK1/2 and STAT1/3 signaling in human PTC. 10 In summary, the final functional cellular outcome of differential NRP isoform expression in renal cells such as tubular epithelial cells or tubulointerstitial fibroblasts could be controlled by gradients of extracellular ligands, by coexpression of signaling receptors, such as plexins or VEGFRs, and by ligands acting through NRPs and their co-receptors.…”

Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K- ras oncogene in vivo

“…Thus, the effects of K-ras mutation may be less aggressive in tissues (such as the intestinal epithelium) in which these pathways are normally activated and where the appropriate feedback loops are expressed. In this light it is of interest to note that within Madin Darby canine kidney cells loss of active ERK͞MEK signaling restores cells to an epithelial state (26).…”