Yannick D. Benoit

Integrin α8β1 regulates adhesion, migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown.

Integrin-linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin-dependent mechanism

Integrin‐linked kinase (ILK) plays a role in integrin signaling‐mediated extracellular matrix (ECM)–cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold‐function interacting partners, PINCH‐1, α‐parvin, and β‐parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non‐proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH‐1, α‐parvin, and β‐parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown‐induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt–villus axis homeostasis—especially with regard to basement membrane fibronectin deposition—as well as cell proliferation, spreading, and migration. J. Cell. Physiol. 222: 387–400, 2010.

Collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions

Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial-stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell-fibronectin interactions.

Cooperation between HNF-1α, Cdx2, and GATA-4 in initiating an enterocytic differentiation program in a normal human intestinal epithelial progenitor cell line

In the intestinal epithelium, the Cdx, GATA, and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx2, GATA-4, and HNF-1alpha using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx2 or HNF-1alpha, alone or in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1alpha and GATA-4 together induced morphological modifications of the cells toward polarization, resulting in the appearance of functional features such as microvilli. HNF-1alpha was also sufficient to induce the expression of cadherins and dipeptidylpeptidase, whereas in combination with Cdx2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large-scale analysis of gene expression confirmed the cooperative effect of these factors. Finally, although DcamKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi1, were unaffected. These observations show that, in cooperation with Cdx2, HNF-1alpha acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 appears to promote morphological changes.

Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells

Colorectal cancer is among the leading causes of cancer death in the USA. The polycomb repressive complex 2 (PRC2), including core components SUZ12 and EZH2, represents a key epigenetic regulator of digestive epithelial cell physiology and was previously shown to promote deleterious effects in a number of human cancers, including colon. Using colon cancer stem cells (CCSC) isolated from human primary colorectal tumors, we demonstrate that SUZ12 knockdown and treatment with DZNep, one of the most potent EZH2 inhibitors, increase apoptosis levels, marked by decreased Akt phosphorylation, in CCSCs, while embryonic stem (ES) cell survival is not affected. Moreover, DZNep treatments lead to increased PTEN expression in these highly tumorigenic cells. Taken together, our findings suggest that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new, epigenetic therapeutic strategy to target highly tumorigenic and metastatic colon cancer stem cells.

Polycomb repressive complex 2 impedes intestinal cell terminal differentiation

Summary The crypt–villus axis constitutes the functional unit of the small intestine, where mature absorptive cells are confined to the villi, and stem cells and transit amplifying and differentiating cells are restricted to the crypts. The polycomb group (PcG) proteins repress differentiation and promote self-renewal in embryonic stem cells. PcGs prevent transcriptional activity by catalysing epigenetic modifications, such as the covalent addition of methyl groups on histone tails, through the action of the polycomb repressive complex 2 (PRC2). Although a role for PcGs in the preservation of stemness characteristics is now well established, recent evidence suggests that they may also be involved in the regulation of differentiation. Using intestinal epithelial cell models that recapitulate the enterocytic differentiation programme, we generated a RNAi-mediated stable knockdown of SUZ12, which constitutes a cornerstone for PRC2 assembly and functionality, in order to analyse intestinal cell proliferation and differentiation. Expression of SUZ12 was also investigated in human intestinal tissues, revealing the presence of SUZ12 in most proliferative epithelial cells of the crypt and an increase in its expression in colorectal cancers. Moreover, PRC2 disruption led to a significant precocious expression of a number of terminal differentiation markers in intestinal cell models. Taken together, our data identified a mechanism whereby PcG proteins participate in the repression of the enterocytic differentiation program, and suggest that a similar mechanism exists in situ to slow down terminal differentiation in the transit amplifying cell population.

Vitamin A Deficiency Causes Hyperglycemia and Loss of Pancreatic β-Cell Mass

Background: Little is known about vitamin A (VA) regulation of pancreatic endocrine mass in adults. Results: Decreased pancreatic VA causes increased α-cell to β-cell mass ratios, hyperglycemia, and hyperglucagonemia. Reintroducing dietary VA restores normoglycemia and α-cell to β-cell mass. Conclusion: VA is essential for maintenance of β-cell functions in adult pancreas. Significance: VA therapies may potentially prevent β-cell apoptosis and loss in diabetes. We show that vitamin A (all-trans-retinol) (VA) is required both for the maintenance of pancreatic β-cell and α-cell mass and for glucose-stimulated insulin secretion in adult mice. Dietary VA deprivation (VAD) causes greatly decreased pancreatic VA levels, hyperglycemia, and reduced insulin secretion. Adult mice fed VAD diets display remodeling of the endocrine pancreas, marked β-cell apoptosis, shifts to smaller islet size distributions, decreased β-cell mass, increased α-cell mass, and hyperglucagonemia. Importantly, although we induced VAD in the entire animal, the pancreatic β-cells are exquisitely sensitive to VAD-associated apoptosis compared with other cell types in other organs. VAD causes major reductions in levels of the VA intracellular binding protein Crbp1 and the retinoic acid-metabolizing enzyme Cyp26a1 specifically in larger islets, suggesting the use of these proteins as biomarkers for early endocrine mass abnormalities. In the VAD mice, the reductions in pancreatic islet sizes and the associated aberrant endocrine functions, which show similarities to the phenotype in advanced type 2 diabetes, result from reductions in pancreatic VA signaling. Reintroduction of dietary VA to VAD mice restores pancreatic VA levels, glycemic control, normal islet size distributions, β-cell to α-cell ratios, endocrine hormone profiles, and RARβ2 and RARγ2 transcript levels. Restoration of β-cell mass by reintroducing VA to VAD mice does not involve increased β-cell proliferation or neogenesis. Pharmacologic modulation of pancreatic VA signaling should be explored for the preservation and/or restoration of pancreatic β-cell mass and function in individuals with diabetes mellitus.

Inhibition of PRC2 histone methyltransferase activity increases TRAIL-mediated apoptosis sensitivity in human colon cancer cells.

Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins, including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation‐associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of retinoic acid receptor β silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3‐deazaneplanocin A (DZNep), we increased TRAIL‐mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ∼2.5‐fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of Polycomb repressive complex 2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non‐responsiveness in a subset of colorectal tumors by increasing TRAIL‐mediated apoptosis sensitivity. J. Cell. Physiol. 228: 764–772, 2013.

Polycomb recruitment attenuates retinoic acid-induced transcription of the bivalent NR2F1 gene.

Polycomb proteins play key roles in mediating epigenetic modifications that occur during cell differentiation. The Polycomb repressive complex 2 (PRC2) mediates the tri-methylation of histone H3 lysine 27 (H3K27me3). In this study, we identify a distinguishing feature of two classes of PRC2 target genes, represented by the Nr2F1 (Coup-TF1) and the Hoxa5 gene, respectively. Both genes are transcriptionally activated by all-trans retinoic acid (RA) and display increased levels of the permissive H3K9/K14ac and tri-methylated histone H3 lysine 4 epigenetic marks in response to RA. However, while in response to RA the PRC2 and H3K27me3 marks are greatly decreased at the Hoxa5 promoter, these marks are initially increased at the Nr2F1 promoter. Functional depletion of the essential PRC2 protein Suz12 by short hairpin RNA (shRNA) technology enhanced the RA-associated transcription of Nr2F1, Nr2F2, Meis1, Sox9 and BMP2, but had no effect on the Hoxa5, Hoxa1, Cyp26a1, Cyp26b1 and RARβ2 transcript levels in wild-type embryonic stem cells. We propose that PRC2 recruitment attenuates the RA-associated transcriptional activation of a subset of genes. Such a mechanism would permit the fine-tuning of transcriptional networks during differentiation.

Deletion of retinoic acid receptor β (RARβ) impairs pancreatic endocrine differentiation.

All-trans retinoic acid (RA) signals via binding to retinoic acid receptors (RARs α, β, and γ). RA directly influences expression of Pdx1, a transcription factor essential for pancreatic development and beta-cell (β-cell) maturation. In this study we follow the differentiation of cultured wild-type (WT) vs. RARβ knockout (KO) embryonic stem (ES) cells into pancreatic islet cells. We found that RARβ KO ES cells show greatly reduced expression of some important endocrine markers of differentiated islet cells, such as glucagon, islet amyloid polypeptide (Iapp), and insulin 1 (Ins1) relative to WT. We conclude that RARβ activity is essential for proper differentiation of ES cells to pancreatic endocrine cells.