title: Constitutive activation of the MEK/ERK pathway inhibits intestinal epithelial cell differentiation.
abstract: The Ras/Raf/MEK/ERK cascade regulates intestinal epithelial cell proliferation. Indeed, while barely detectable in differentiated cells of the villi, ERK1/2-activated forms are detected in the nucleus of undifferentiated human intestinal crypt cells. In addition, we and others have reported that ERKs are selectively inactivated during enterocyte differentiation. However, whether inactivation of the ERK pathway is necessary for inhibition of both proliferation and induction of differentiation of intestinal epithelial cells is unknown. Human Caco-2/15 cells, undifferentiated crypt IEC-6 cells, and differentiating Cdx3-expressing IEC-6 cells were infected with retroviruses encoding either a hemagglutinin (HA)-tagged MEK1 wild type (wtMEK) or a constitutively active S218D/S222D MEK1 mutant (caMEK). Protein and gene expression was assessed by Western blotting, semiquantitative RT-PCR, and real-time PCR. Morphology was analyzed by transmission electron microscopy. We found that 1) IEC-6/Cdx3 cells formed multicellular layers after confluence and differentiated after 30 days in culture, as assessed by increased polarization, microvilli formation, expression of differentiation markers, and ERK1/2 inhibition; 2) while activated MEK prevented neither the inhibition of ERK1/2 activities nor the differentiation process in postconfluent Caco-2/15 cells, caMEK expression prevented ERK inhibition in postconfluent IEC-6/Cdx3 cells, thus leading to maintenance of elevated ERK1/2 activities; 3) caMEK-expressing IEC-6/Cdx3 cells exhibited altered multicellular structure organization, poorly defined tight junctions, reduced number of microvilli on the apical surface, and decreased expression of the hepatocyte nuclear factor 1α transcription factor and differentiation markers, namely apolipoprotein A-4, fatty acid-binding protein, calbindin-3, mucin 2, alkaline phosphatase, and sucrase-isomaltase; and 4) increased Cdx3 phosphorylation on serine-60 (S60) in IEC-6/Cdx3 cells expressing caMEK led to decreased Cdx2 transactivation potential. These results indicate that inactivation of the ERK pathway is required to ensure the full Cdx2/3 transcriptional activity necessary for intestinal epithelial cell terminal differentiation.
authors: Canadian Institutes of Health Research Team on Digestive Epithelium, Department of Anatomy and Cellular Biology, Quebec.
authors: Etienne
authors: E
authors: Lemieux
authors: Marie-Josée
authors: MJ
authors: Boucher
authors: Sébastien
authors: S
authors: Mongrain
authors: François
authors: F
authors: Boudreau
authors: Claude
authors: C
authors: Asselin
authors: Nathalie
authors: N
authors: Rivard
mesh_terms: D000818:Animals; D000071616:CDX2 Transcription Factor; D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D018398:Homeodomain Proteins / Q000502:physiology; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D051381:Rats; D015534:Trans-Activators / Q000502:physiology
chemical_list: D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; D018398:Homeodomain Proteins; D015534:Trans-Activators; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases