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.

Deficiency of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Intestinal Epithelial Cells Has No Appreciable Impact on Dextran Sulphate Sodium Colitis Severity But Promotes Wound Healing

“…EGFR is among the known targets of PTPN2 [5] , which might explain the increased proliferation in PTPN2-deficient IEC. Also ERK and p38 -both targets of PTPN2 in IEC [23,25] -have been implicated in IEC proliferation [26,27] . However, further analysis would be necessary to address the molecular mechanisms as to how loss of PTPN2 mediates increased proliferation.…”

Generation of Enterocyte-Like Cells with Pharmacokinetic Functions from Human Induced Pluripotent Stem Cells Using Small-Molecule Compounds

“…In particular, PD98059, 5-aza-29-deoxycytidine, and A-83-01 were useful in enhancing the differentiation and gain of pharmacokinetic function. The constitutive activation of mitogen-activated protein/extracellular signal-regulated kinase led to decreased caudal type homeobox 2, a major transcription factor associated with intestinal development and differentiation, and resulted in the inhibition of intestinal differentiation (Lemieux et al, 2011). We accordingly speculated that PD98059, which is a mitogen-activated protein inhibitor, promoted intestinal differentiation through this pathway.…”

Epithelial Tyrosine Phosphatase SHP-2 Protects against Intestinal Inflammation in Mice

“…The human colon cell line Caco-2/15, obtained from Andrea Quaroni (Cornell University, Ithaca, NY), and the rat intestinal epithelial cell line IEC-6, provided by the ATCC (Manassas, VA), were cultured as described previously (22). IEC-6 cells were serum starved for 24 h and then treated with 10 ng/ml of interleukin 6 (IL-6) (Bioshop, Burlington, ON, Canada) or 12.5 g/ml of lipopolysaccharide (LPS) (serotype O111:B4; Enzo Life Sciences, Farmingdale, NY).…”