Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn's disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

The transcription factor hepatocyte nuclear factor 4A acts in the intestine to promote white adipose tissue energy storage.

The transcription factor hepatocyte nuclear factor 4 A (HNF4A) controls the metabolic features of several endodermal epithelia. Both HNF4A and HNF4G are redundant in the intestine and it remains unclear whether HNF4A alone controls intestinal lipid metabolism. Here we show that intestinal HNF4A is not required for intestinal lipid metabolism per se, but unexpectedly influences whole-body energy expenditure in diet-induced obesity (DIO). Deletion of intestinal HNF4A caused mice to become DIO-resistant with a preference for fat as an energy substrate and energetic changes in association with white adipose tissue (WAT) beiging. Intestinal HNF4A is crucial for the fat-induced release of glucose-dependent insulinotropic polypeptide (GIP), while the reintroduction of a stabilized GIP analog rescues the DIO resistance phenotype of the mutant mice. Our study provides evidence that intestinal HNF4A plays a non-redundant role in whole-body lipid homeostasis and points to a non-cell-autonomous regulatory circuit for body-fat management.

Comprehensive Profiling of Early Neoplastic Gastric Microenvironment Modifications and Biodynamics in Impaired BMP-Signaling FoxL1+-Telocytes.

FoxL1+telocytes (TCFoxL1+) are novel gastrointestinal subepithelial cells that form a communication axis between the mesenchyme and epithelium. TCFoxL1+ are strategically positioned to be key contributors to the microenvironment through production and secretion of growth factors and extracellular matrix (ECM) proteins. In recent years, the alteration of the bone morphogenetic protein (BMP) signaling in TCFoxL1+ was demonstrated to trigger a toxic microenvironment with ECM remodeling that leads to the development of pre-neoplastic gastric lesions. However, a comprehensive analysis of variations in the ECM composition and its associated proteins in gastric neoplasia linked to TCFoxL1+ dysregulation has never been performed. This study provides a better understanding of how TCFoxL1+ defective BMP signaling participates in the gastric pre-neoplastic microenvironment. Using a proteomic approach, we determined the changes in the complete matrisome of BmpR1a△FoxL1+ and control mice, both in total antrum as well as in isolated mesenchyme-enriched antrum fractions. Comparative proteomic analysis revealed that the deconstruction of the gastric antrum led to a more comprehensive analysis of the ECM fraction of gastric tissues microenvironment. These results show that TCFoxL1+ are key members of the mesenchymal cell population and actively participate in the establishment of the matrisomic fraction of the microenvironment, thus influencing epithelial cell behavior.

Altered Mucus Barrier Integrity and Increased Susceptibility to Colitis in Mice upon Loss of Telocyte Bone Morphogenetic Protein Signalling.

FoxL1+-Telocytes (TCFoxL1+) are subepithelial cells that form a network underneath the epithelium. We have shown that without inflammatory stress, mice with loss of function in the BMP signalling pathway in TCFoxL1+ (BmpR1aΔFoxL1+) initiated colonic neoplasia. Although TCFoxL1+ are modulated in IBD patients, their specific role in this pathogenesis remains unclear. Thus, we investigated how the loss of BMP signalling in TCFoxL1+ influences the severity of inflammation and fosters epithelial recovery after inflammatory stress. BmpR1a was genetically ablated in mouse colonic TCFoxL1+. Experimental colitis was performed using a DSS challenge followed by recovery steps to assess wound healing. Physical barrier properties, including mucus composition and glycosylation, were assessed by alcian blue staining, immunofluorescences and RT-qPCR. We found that BmpR1aΔFoxL1+ mice had impaired mucus quality, and upon exposure to inflammatory challenges, they had increased susceptibility to experimental colitis and delayed healing. In addition, defective BMP signalling in TCFoxL1+ altered the functionality of goblet cells, thereby affecting mucosal structure and promoting bacterial invasion. Following inflammatory stress, TCFoxL1+ with impaired BMP signalling lose their homing signal for optimal distribution along the epithelium, which is critical in tissue regeneration after injury. Overall, our findings revealed key roles of BMP signalling in TCFoxL1+ in IBD pathogenesis.

NCOR1 Sustains Colorectal Cancer Cell Growth and Protects against Cellular Senescence.

NCOR1 is a corepressor that mediates transcriptional repression through its association with nuclear receptors and specific transcription factors. Some evidence supports a role for NCOR1 in neonatal intestinal epithelium maturation and the maintenance of epithelial integrity during experimental colitis in mice. We hypothesized that NCOR1 could control colorectal cancer cell proliferation and tumorigenicity. Conditional intestinal epithelial deletion of Ncor1 in ApcMin/+ mice resulted in a significant reduction in polyposis. RNAi targeting of NCOR1 in Caco-2/15 and HT-29 cell lines led to a reduction in cell growth, characterized by cellular senescence associated with a secretory phenotype. Tumor growth of HT-29 cells was reduced in the absence of NCOR1 in the mouse xenografts. RNA-seq transcriptome profiling of colon cancer cells confirmed the senescence phenotype in the absence of NCOR1 and predicted the occurrence of a pro-migration cellular signature in this context. SOX2, a transcription factor essential for pluripotency of embryonic stem cells, was induced under these conditions. In conclusion, depletion of NCOR1 reduced intestinal polyposis in mice and caused growth arrest, leading to senescence in human colorectal cell lines. The acquisition of a pro-metastasis signature in the absence of NCOR1 could indicate long-term potential adverse consequences of colon-cancer-induced senescence.

HNF4α is a novel regulator of intestinal glucose-dependent insulinotropic polypeptide.

Mutations in the HNF4A gene cause MODY1 and are associated with an increased risk of Type 2 diabetes mellitus. On the other hand, incretins are hormones that potentiate reductions in blood glucose levels. Given the established role of incretin-based therapy to treat diabetes and metabolic disorders, we investigated a possible regulatory link between intestinal epithelial HNF4α and glucose-dependent insulinotropic polypeptide (GIP), an incretin that is specifically produced by gut enteroendocrine cells. Conditional deletion of HNF4α in the whole intestinal epithelium was achieved by crossing Villin-Cre and Hnf4αloxP/loxP C57BL/6 mouse models. GIP expression was measured by qPCR, immunofluorescence and ELISA. Gene transcription was assessed by luciferase and electrophoretic mobility shift assays. Metabolic parameters were analyzed by indirect calorimetry and dual-energy X-ray absorptiometry. HNF4α specific deletion in the intestine led to a reduction in GIP. HNF4α was able to positively control Gip transcriptional activity in collaboration with GATA-4 transcription factor. Glucose homeostasis and glucose-stimulated insulin secretion remained unchanged in HNF4α deficient mice. Changes in GIP production in these mice did not impact nutrition or energy metabolism under normal physiology but led to a reduction of bone area and mineral content, a well described physiological consequence of GIP deficiency. Our findings point to a novel regulatory role between intestinal HNF4α and GIP with possible functional impact on bone density.

Transcription factor CUX1 is required for intestinal epithelial wound healing and targets the VAV2-RAC1 Signalling complex.

Intestinal epithelial cells form a protective barrier in limiting gut luminal content potentially harmful to the host. Upon gut epithelium injury, several signals instruct epithelial cells to undergo a rapid healing process. Defects in this process induce inflammatory responses and can further evolve into chronic gut inflammatory diseases. We previously identified the transcription factor CUX1 as crucial for protecting against experimental colitis in mice. However, the precise molecular mechanisms by which CUX1 intervenes during this biological process are unknown. Our aim was to evaluate CUX1 biological and functional roles during intestinal epithelial cell wound healing. RNAi knockdown of CUX1 in intestinal epithelial cells revealed a crucial role for this regulator in migratory response following wounding assays. Gene expression profiling identified several gene transcripts modulated in absence of CUX1 during wound healing for which a significant number was associated with cell motility and cytoskeleton function. Chromatin immunoprecipitation assays identified the guanine nucleotide exchange factor Vav2 gene as a direct target for CUX1. Coincidently, reduction of VAV2 in absence of CUX1 was associated with a significant decrease of RAC1 activity in response to epithelial wounding. Our results identify a novel pathway by which CUX1 regulates normal intestinal epithelial cell restitution.

Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade.

Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1a(ΔMES)). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1a(ΔMES) stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation.

Bmp signaling in colonic mesenchyme regulates stromal microenvironment and protects from polyposis initiation.

In the colon, myofibroblasts are primary contributors in the establishment of the microenvironment involved in tissue homeostasis. Alterations in myofibroblast functions lead to changes resulting in a toxic microenvironment nurturing tumorigenesis. Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in adult gut homeostasis. Studies in genetically-modified mice have shown that Bmp disruption in all cell layers leads to the development of gut polyposis. In contrast, our studies showed that loss of Bmp exclusively in the gastrointestinal epithelium resulted in increased epithelial proliferation without polyposis initiation, thus suggesting a key role for mesenchymal Bmp signaling in polyposis initiation. In order to identify the role of mesenchymal Bmp signaling on the microenvironment and its impact on colonic mucosa, a mouse model was generated with suppression of Bmp signaling exclusively in myofibroblasts (Bmpr1aΔMES). Bmpr1aΔMES mice exhibited increased subepithelial proliferation with changes in cellular composition leading to the development of a primed stroma with modulation of extracellular matrix proteins, immune cells and cytokines as early as 90 days of age. This microenvironmental deregulation was associated with increased polyposis initiation at one year of age. These results are the first to demonstrate that mesenchymal Bmpr1a inactivation alone is sufficient to prompt an expansion of myofibroblasts leading to the development of a reactive mesenchyme that contributes to polyposis initiation in the colon. These findings support the novel concept that inhibition of Bmp signaling in mesenchymal cells surrounding the normal epithelium leads to important changes instructing a toxic microenvironment sufficient to induce colonic polyposis.

Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis.

The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin-Cre-inducible intestinal epithelial cell (IEC)-specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin-Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short-term deletion of both genes in naphtoflavone-inducible Ah-Cre and tamoxifen-inducible villin-Cre(ER) mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS-induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS-induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR, and Stat3 signaling pathways was observed. While villin-Cre(ER) Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah-Cre-mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment.

HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation.

Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation.

Epithelial tyrosine phosphatase SHP-2 protects against intestinal inflammation in mice.

Polymorphisms of PTPN11 encoding SHP-2 are biomarkers for ulcerative colitis (UC) susceptibility. However, their functional relevance is unknown. We thus investigated the role of epithelial SHP-2 in the control of intestinal homeostasis. Mice with an intestinal epithelial cell-specific SHP-2 deletion (SHP-2(IEC-KO) mice) were generated. Control and SHP-2(IEC-KO) mice were monitored for clinical symptoms and sacrificed for histological staining and Western blot analyses. Cytokines and chemokines, as well as intestinal permeability, were quantified. SHP-2 mRNA expression was evaluated in control and UC patients. SHP-2(IEC-KO) mice showed growth retardation compared to control littermates and rapidly developed severe colitis. Colon architecture was markedly altered with infiltration of immune cells, crypt abscesses, neutrophil accumulation, and reduced goblet cell numbers. Decreased expression of claudins was associated with enhanced intestinal permeability in mutant SHP-2(IEC-KO) mice. Inflammatory transcription factors Stat3 and NF-κB were hyperactivated early in the mutant colonic epithelium. Levels of several epithelial chemokines and cytokines were markedly enhanced in SHP-2(IEC-KO) mice. Of note, antibiotic treatment remarkably impaired the development of colitis in SHP-2(IEC-KO) mice. Finally, SHP-2 mRNA levels were significantly reduced in intestinal biopsy specimens from UC patients. Our results establish intestinal epithelial SHP-2 as a critical determinant for prevention of gut inflammation.

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.

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells.

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis.

The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5(ΔIEC) and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5(ΔIEC) mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohn's disease and ulcerative colitis samples. Smad5(ΔIEC) mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing.

Hepatocyte nuclear factor-4alpha promotes gut neoplasia in mice and protects against the production of reactive oxygen species.

Hepatocyte nuclear factor-4α (Hnf4α) is a transcription factor that controls epithelial cell polarity and morphogenesis. Hnf4α conditional deletion during postnatal development has minor effects on intestinal epithelium integrity but promotes activation of the Wnt/ß-catenin pathway without causing tumorigenesis. Here, we show that Hnf4α does not act as a tumor-suppressor gene but is crucial in promoting gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice lacking Hnf4α is suppressed compared with littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4α in the intestinal epithelium identifies novel functions of this transcription factor in targeting oxidoreductase-related genes involved in the regulation of reactive oxygen species (ROS) levels. This role is supported with the demonstration that HNF4α is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of ROS in colorectal cancer cell lines. Analysis of a colorectal cancer patient cohort establishes that HNF4α is significantly upregulated compared with adjacent normal epithelial resections. Several genes involved in ROS neutralization are also induced in correlation with HNF4A expression. Altogether, the findings point to the nuclear receptor HNF4α as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis.

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.

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 beta-catenin protein levels, nor is it sufficient to promote tumorigenesis initiation. However, it severely enhances intestinal tumor load in Apc(Min/+) 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.