Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

Transcription factor achaete scute-like 2 controls intestinal stem cell fate.

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development.

In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.

Stem cells, self-renewal, and differentiation in the intestinal epithelium.

The mammalian intestine is covered by a single layer of epithelial cells that is renewed every 4-5 days. This high cell turnover makes it a very attractive and comprehensive adult organ system for the study of cell proliferation and differentiation. The intestine is composed of proliferative crypts, which contain intestinal stem cells, and villi, which contain differentiated specialized cell types. Through the recent identification of Lgr5, an intestinal stem cell marker, it is now possible to visualize stem cells and study their behavior and differentiation in a much broader context. In this review we describe the identification of intestinal stem cells. We also discuss genetic studies that have helped to elucidate those signals important for progenitor cells to differentiate into one of the specialized intestinal epithelial cell types. These studies describe a genetic hierarchy responsible for cell fate commitment in normal gut physiology. Where relevant we also mention aberrant deregulation of these molecular pathways that results in colon cancer.

Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro.

BACKGROUND: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8. METHODS: IL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-alpha, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-(14)C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation. RESULTS: For both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-alpha-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively. CONCLUSION: We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

Transcriptome profile of human colorectal adenomas.

Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer.

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.

Ascl2 acts as an R-spondin/Wnt-responsive switch to control stemness in intestinal crypts.

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and in many other adult organs. The transcription factor Ascl2 (a Wnt target gene) is a master regulator of intestinal stem cell identity. It is unclear how the continuous Wnt gradient along the crypt axis is translated into discrete expression of Ascl2 and discrete specification of stem cells at crypt bottoms. We show that (1) Ascl2 is regulated in a direct autoactivatory loop, leading to a distinct on/off expression pattern, and (2) Wnt/R-spondin can activate this regulatory loop. This mechanism interprets the Wnt levels in the intestinal crypt and translates the continuous Wnt signal into a discrete Ascl2 "on" or "off" decision. In turn, Ascl2, together with ß-catenin/Tcf, activates the genes fundamental to the stem cell state. In this manner, Ascl2 forms a transcriptional switch that is both Wnt responsive and Wnt dependent to define stem cell identity.

Robust cre-mediated recombination in small intestinal stem cells utilizing the olfm4 locus.

The epithelium of the small intestine is the most rapidly self-renewing tissue in mammals. We previously demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression at the bottom of intestinal crypts. An Lgr5-eGFP-IRES-CreERT2 knockin allele has been instrumental in characterizing and profiling these cells, yet its low level expression and its silencing in patches of adjacent crypts have not allowed quantitative gene deletion. Olfactomedin-4 (Olfm4) has emerged from a gene signature of Lgr5 stem cells as a robust marker for murine small intestinal stem cells. We observe that Olfm4(null) animals show no phenotype and report the generation of an Olfm4-IRES-eGFPCreERT2 knockin mouse model that allows visualization and genetic manipulation of Lgr5+ stem cells in the epithelium of the small intestine. The eGFPCreERT2 fusion protein faithfully marks all stem cells in the small intestine and induces the activation of a conditional LacZ reporter with robust efficiency.

Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells.

Wnt signaling activates gene expression through the induced formation of complexes between DNA-binding T-cell factors (TCFs) and the transcriptional coactivator beta-catenin. In colorectal cancer, activating Wnt pathway mutations transform epithelial cells through the inappropriate activation of a TCF7L2/TCF4 target gene program. Through a DNA array-based genome-wide analysis of TCF4 chromatin occupancy, we have identified 6,868 high-confidence TCF4-binding sites in the LS174T colorectal cancer cell line. Most TCF4-binding sites are located at large distances from transcription start sites, while target genes are frequently "decorated" by multiple binding sites. Motif discovery algorithms define the in vivo-occupied TCF4-binding site as evolutionarily conserved A-C/G-A/T-T-C-A-A-A-G motifs. The TCF4-binding regions significantly correlate with Wnt-responsive gene expression profiles derived from primary human adenomas and often behave as beta-catenin/TCF4-dependent enhancers in transient reporter assays.

The Intestinal Wnt/TCF Signature.

BACKGROUND & AIMS: In colorectal cancer, activating mutations in the Wnt pathway transform epithelial cells through the inappropriate expression of a TCF4 target gene program, which is physiologically expressed in intestinal crypts. METHODS: We have now performed an exhaustive array-based analysis of this target gene program in colorectal cancer cell lines carrying an inducible block of the Wnt cascade. Independently, differential gene-expression profiles of human adenomas and adenocarcinomas vs normal colonic epithelium were obtained. RESULTS: Expression analyses of approximately 80 genes common between these data sets were performed in a murine adenoma model. The combined data sets describe a core target gene program, the intestinal Wnt/TCF signature gene set, which is responsible for the transformation of human intestinal epithelial cells. CONCLUSIONS: The genes were invariably expressed in adenomas, yet could be subdivided into 3 modules, based on expression in distinct crypt compartments. A module of 17 genes was specifically expressed at the position of the crypt stem cell.

Mass-dependent decline of skeletal muscle function in cancer cachexia.

CD2F1 mice were inoculated with C26 adenocarcinoma cells, followed by assessment of ex vivo muscular function. Muscles from tumor-bearing mice had a significantly lower force output during a single maximal contraction and during repeated contractions than control muscles. The relative force output, however, did not differ when corrected for muscle mass. Thus, cachexia significantly reduces absolute skeletal muscle function, but muscle "quality" appears unaltered.