Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo

BCL9/9L-β-catenin Signaling is Associated With Poor Outcome in Colorectal Cancer

BCL9/9L proteins enhance the transcriptional output of the β-catenin/TCF transcriptional complex and contribute critically to upholding the high WNT signaling level required for stemness maintenance in the intestinal epithelium. Here we show that a BCL9/9L-dependent gene signature derived from independent mouse colorectal cancer (CRC) models unprecedentedly separates patient subgroups with regard to progression free and overall survival. We found that this effect was by and large attributable to stemness related gene sets. Remarkably, this signature proved associated with recently described poor prognosis CRC subtypes exhibiting high stemness and/or epithelial-to-mesenchymal transition (EMT) traits. Consistent with the notion that highWNT signaling is required for stemness maintenance, ablating Bcl9/9l-β-catenin in murine oncogenic intestinal organoids provoked their differentiation and completely abrogated their tumorigenicity, while not affecting their proliferation. Therapeutic strategies aimed at targeting WNT responses may be limited by intestinal toxicity. Our findings suggest that attenuating WNT signaling to an extent that affects stemness maintenance without disturbing intestinal renewal might be well tolerated and prove sufficient to reduce CRC recurrence and dramatically improve disease outcome

β-catenin negatively regulates expression of the prostaglandin transporter PGT in the normal intestinal epithelium and colorectal tumour cells: A role in the chemopreventive efficacy of aspirin

Background: Levels of the pro-tumorigenic prostaglandin PGE 2 are increased in colorectal cancer, previously attributed to increased synthesis through COX-2 upregulation and, more recently, to decreased catabolism. The functionally linked genes 15-prostaglandin dehydrogenase (15-PGDH) and the prostaglandin transporter PGT co-operate in prostaglandin degradation and are downregulated in colorectal cancer. We previously reported repression of 15-PGDH expression by the Wnt/β-catenin pathway, commonly deregulated during early colorectal neoplasia. Here we asked whether β-catenin also regulates PGT expression. Methods: The effect of β-catenin deletion in vivo was addressed by PGT immunostaining of β-catenin/lox-villin-cre-ERT2 mouse tissue. The effect of siRNA-mediated β-catenin knockdown and dnTCF4 induction in vitro was addressed by semi-quantitative and quantitative real-time RT-PCR and immunoblotting. Results: This study shows for the first time that deletion of β-catenin in murine intestinal epithelium in vivo upregulates PGT protein, especially in the crypt epithelium. Furthermore, β-catenin knockdown in vitro increases PGT expression in both colorectal adenoma-and carcinoma-derived cell lines, as does dnTCF4 induction in LS174T cells.Conclusions:These data suggest that β-catenin employs a two-pronged approach to inhibiting prostaglandin turnover during colorectal neoplasia by repressing PGT expression in addition to 15-PGDH. Furthermore, our data highlight a potential mechanism that may contribute to the non-selective NSAID aspirins chemopreventive efficacy. © 2012 Cancer Research UK All rights reserved

SOX2 redirects the developmental fate of the intestinal epithelium toward a premature gastric phenotype

__Abstract__ Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut

A Key Role for E-cadherin in Intestinal Homeostasis and Paneth Cell Maturation

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells

Modelling the Spatio-Temporal Cell Dynamics Reveals Novel Insights on Cell Differentiation and Proliferation in the Small Intestinal Crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4–5 days with 10–12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days

Gradients in the in vivo intestinal stem cell compartment and their in vitro recapitulation in mimetic platforms

peer-reviewedIntestinal tissue, and specifically its mucosal layer, is a complex and gradient-rich environment. Gradients of soluble factor (BMP, Noggin, Notch, Hedgehog, and Wnt), insoluble extracellular matrix proteins (laminins, collagens, fibronectin, and their cognate receptors), stromal stiffness, oxygenation, and sheer stress induced by luminal fluid flow at the crypt-villus axis controls and supports healthy intestinal tissue homeostasis. However, due to current technological challenges, very few of these features have so far been included in in vitro intestinal tissue mimetic platforms. In this review, the tightly defined and dynamic microenvironment of the intestinal tissue is presented in detail. Additionally, the authors introduce the current state-of-the-art intestinal tissue mimetic platforms, as well as the design drawbacks and challenges they face while attempting to capture the complexity of the intestinal tissue’s physiology. Finally, the compositions of an “idealized” mimetic system is presented to guide future developmental efforts

Völkisch und sozial? : Neonazistische Agitation gegen die neue EU-Freizügigkeit für Arbeitnehmerinnen

Wnt/β-catenin signalling pathway is crucial for the formation of many tissues and organs during development. In recent years, this pathway has also been found to regulate the biology of stem cells in the intestine and probably in other organs in adult life. Abnormal activation of Wnt/β-catenin signalling, which controls the expression of a high number of genes, is critical for the initiation and progression of most colorectal cancers. In line with this, the gene expression signature induced by activation of the Wnt/β-catenin pathway defines the intestinal stem cells present at the bottom of the crypts and also colon cancer stem cells. This supports the importance of inhibitors of the Wnt/β-catenin pathway as potential agents in colorectal cancer therapy. However, the complexity, wide activity in the organism modulating the biology of several cell types, and characteristics of this pathway have delayed the identification of suitable targets and so, the development of such inhibitors that are only now reaching the clinic.Peer reviewe

Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE