Marie-Josée Langlois

Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells

In vitro experiments have shown that the establishment of cell–cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E‐cadherin‐mediated cell–cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco‐2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E‐cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK–ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 μM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K‐dependent manner; (4) adenoviral infection of confluent Caco‐2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK–ERK1/2 in confluent differentiating Caco‐2/15 cells, but not in undifferentiated growing Caco‐2/15 cells. Our data suggest that E‐cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt‐dependent pathway. This mechanism may account for the role of E‐cadherin in proliferation/differentiation transition along the crypt‐villus axis of the human intestinal epithelium. J. Cell. Physiol. 199: 32–39, 2004© 2003 Wiley‐Liss, Inc.

The PTEN Phosphatase Controls Intestinal Epithelial Cell Polarity and Barrier Function: Role in Colorectal Cancer Progression

Background The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells. Methodology/Principal Findings Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice. Conclusions/Significance Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.

Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non‐permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy.

Epithelial phosphatase and tensin homolog regulates intestinal architecture and secretory cell commitment and acts as a modifier gene in neoplasia

Phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3‐kinase/Akt pathway, is one of the most frequently mutated/deleted tumor suppressor genes in human cancers. The aim of this study was to gain insight into the role played by PTEN in intestinal homeostasis and epithelial cell function. Using the Cre/loxP system, we have generated a mouse with a conditional intestinal epithelial Pten deficiency. Pten mutant mice and controls were sacrificed for histology, immunofluorescence, Western blot, and quantitative polymerase chain reaction analysis. Our results show that loss of epithelial Pten leads to an intestinalomegaly associated with an increase in epithelial cell proliferation. Histological analysis demonstrated significant perturbation of the crypt‐villus architecture, a marked increase in goblet cells and a decrease in enteroendocrine cells, suggesting a role for Pten in the commitment of the multipotential‐secretory precursor cell. Loss of epithelial Pten does not result in induction of nuclear β‐catenin protein levels, nor is it sufficient to promote tumorigenesis initiation. However, it severely enhances intestinal tumor load in ApcMin/+ mice, in which c‐Myc is already deregulated. These results reveal an unknown function for Pten signaling in the commitment of multipotential‐secretory progenitor cells and suggest that epithelial Pten functions as a modifier gene in intestinal neoplasia.—Langlois, M.‐J., Roy, S. A. B., Auclair, B. A., Jones, C., Boudreau, F., Carrier, J. C., Rivard, N., Perreault, N. Epithelial phosphatase and tensin homolog regulates intestinal architecture and secretory cell commitment and acts as a modifier gene in neoplasia. FASEB J. 23, 1835–1844 (2009)

Loss of Hepatocyte-Nuclear-Factor-1α Impacts on Adult Mouse Intestinal Epithelial Cell Growth and Cell Lineages Differentiation

Background and Aims Although Hnf1α is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1α on the maintenance of adult small intestinal epithelial functions. Methodology/Principal Findings An Hnf1α knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1α displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1α mutant mice. The intestinal epithelium of Hnf1α null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. Conclusions/Significance Together, these results unravel a functional role for Hnf1α in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

SHP-2 Phosphatase Prevents Colonic Inflammation by Controlling Secretory Cell Differentiation and Maintaining Host-Microbiota Homeostasis.

Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP‐2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell‐specific deletion of SHP‐2 (SHP‐2IEC‐KO) develop severe colitis 1 month after birth. However, the mechanisms by which SHP‐2 deletion induces colonic inflammation remain to be elucidated. We generated SHP‐2IEC‐KO mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up‐regulated in SHP‐2‐deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP‐2IEC‐KO mice whereas Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed 2 weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP‐2IEC‐KO mice, rescued Goblet/intermediate cell ratio, and prevented NFκB hyperactivation and inflammation. These data indicate that SHP‐2 is functionally important for the maintenance of appropriate barrier function and host‐microbiota homeostasis in the large intestine. J. Cell. Physiol. 231: 2529–2540, 2016.

The MEK2-binding tumor suppressor hDlg is recruited by E-cadherin to the midbody ring

BackgroundThe human homologue of the Drosophila Discs-large tumor suppressor protein, hDlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. At both synaptic junctions and epithelia cell-cell junctions, hDlg is known to recruit several signaling proteins into macromolecular complexes. hDlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear.ResultsHere we describe the interaction of hDlg with the activated form of MEK2 of the canonical RAF/MEK/ERK pathway, a protein that is found at the midbody during cytokinesis. We show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the PDZ domains of hDlg and the C-terminal portion of MEK2 is dependent on the phosphorylation of MEK2. Finally, we found that E-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hDlg, but not activated MEK2, to that structure.ConclusionOur results suggest that like at other cell-cell junction sites, hDlg is part of a macromolecular complex of structural and signaling proteins at the midbody.

Dual regulatory role for phosphatase and tensin homolog in specification of intestinal endocrine cell subtypes.

AIM To investigate the impact of phosphatase and tensin homolog (Pten) in the specification of intestinal enteroendocrine subpopulations. METHODS Using the Cre/loxP system, a mouse with conditional intestinal epithelial Pten deficiency was generated. Pten mutant mice and controls were sacrificed and small intestines collected for immunofluorescence and quantitative real-time polymerase chain reaction. Blood was collected on 16 h fasted mice by cardiac puncture. Enzyme-linked immunosorbent assay was used to measure blood circulating ghrelin, somatostatin (SST) and glucose-dependent insulinotropic peptide (GIP) levels. RESULTS Results show an unexpected dual regulatory role for epithelial Pten signalling in the specification/differentiation of enteroendocrine cell subpopulations in the small intestine. Our data indicate that Pten positively regulates chromogranin A (CgA) expressing subpopulations, including cells expressing secretin, ghrelin, gastrin and cholecystokinin (CCK). In contrast, Pten negatively regulates the enteroendocrine subtype specification of non-expressing CgA cells such as GIP and SST expressing cells. CONCLUSION The present results demonstrate that Pten signalling favours the enteroendocrine progenitor to specify into cells expressing CgA including those producing CCK, gastrin and ghrelin.

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis: BEAUDRY et al.

The Ras/mitogen‐activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual‐specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal‐regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium‐induced colitis, as opposed to wild‐type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage‐independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.