Guardian crypt-base-goblet-cells protect the human colonic stem cell niche by triggering cholinergic calcium signal-dependent MUC2 secretion and luminal flushing

Mucus secreting goblet cells play a vital role in the maintenance of tissue homeostasis. Here we report the discovery of an enigmatic mechanism for the generation of calcium signals that couple cholinergic input to secretion of hydrated mucus in the human colonic stem cell niche. Mechanistic insights for this study were derived from native human colonic crypts and crypt-like organoids expressing MUC2-mNEON. Importantly, we demonstrate that the human colonic stem cell niche is also a cholinergic niche, and that activation of muscarinic receptors initiates calcium signals at the apical pole of intestinal stem cells and neighbouring crypt-base-goblet-cells. The calcium signal trigger zone is defined by a microdomain of juxtaposed calcium stores expressing TPC1 and InsP3R3 calcium channels. Co-activation of TPC1 and InsP3R3 is required for generation of cholinergic calcium signals and downstream secretion of hydrated mucus, which culminates in the flushing of the colonic stem cell niche.

CCN1 coordinately regulates intestinal stem cell proliferation and differentiation through integrins αvβ3/αvβ5

Intestinal stem cells (ISCs) at the crypt base contribute to intestinal homeostasis through a balance between self-renewal and differentiation. However, the molecular mechanisms regulating this homeostatic balance remain elusive. Here we show that the matricellular protein CCN1/CYR61 coordinately regulates ISC proliferation and differentiation through distinct pathways emanating from CCN1 interaction with integrins αvβ3/αvβ5. Mice that delete Ccn1 in Lgr5+ ISCs or express mutant CCN1 unable to bind integrins αvβ3/αvβ5 exhibited exuberant ISC expansion and enhanced differentiation into secretory cells at the expense of absorptive enterocytes in the small intestine, leading to nutrient malabsorption. Analysis of crypt organoids revealed that through integrins αvβ3/αvβ5, CCN1 induces NF-κB-dependent Jag1 expression to regulate Notch activation for differentiation and promotes Src-mediated YAP activation and Dkk1 expression to control Wnt signaling for proliferation. Moreover, CCN1 and YAP amplify the activities of each other in a regulatory loop. These findings establish CCN1 as a novel niche factor in the intestinal crypts, providing new insights into how matrix signaling exerts overarching control of ISC homeostasis.

Butyrate and the Intestinal Epithelium: Modulation of Proliferation and Inflammation in Homeostasis and Disease

The microbial metabolite butyrate serves as a link between the intestinal microbiome and epithelium. The monocarboxylate transporters MCT1 and SMCT1 are the predominant means of butyrate transport from the intestinal lumen to epithelial cytoplasm, where the molecule undergoes rapid β-oxidation to generate cellular fuel. However, not all epithelial cells metabolize butyrate equally. Undifferentiated colonocytes, including neoplastic cells and intestinal stem cells at the epithelial crypt base preferentially utilize glucose over butyrate for cellular fuel. This divergent metabolic conditioning is central to the phenomenon known as “butyrate paradox”, in which butyrate induces contradictory effects on epithelial proliferation in undifferentiated and differentiated colonocytes. There is evidence that accumulation of butyrate in epithelial cells results in histone modification and altered transcriptional activation that halts cell cycle progression. This manifests in the apparent protective effect of butyrate against colonic neoplasia. A corollary to this process is butyrate-induced inhibition of intestinal stem cells. Yet, emerging research has illustrated that the evolution of the crypt, along with butyrate-producing bacteria in the intestine, serve to protect crypt base stem cells from butyrate’s anti-proliferative effects. Butyrate also regulates epithelial inflammation and tolerance to antigens, through production of anti-inflammatory cytokines and induction of tolerogenic dendritic cells. The role of butyrate in the pathogenesis and treatment of intestinal neoplasia, inflammatory bowel disease and malabsorptive states is evolving, and holds promise for the potential translation of butyrate’s cellular function into clinical therapies.

Protein arginine methyltransferase 1 regulates cell proliferation and differentiation in adult mouse adult intestine

Background Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse. Methods We have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium. Results We show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged. Conclusions Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

Valeric acid-5-HT-dependent macrophage activation drives intestinal stem cell self-renewal

Lgr5+ intestinal stem cells reside within specialized niches at the crypt base and harbor self-renewal and differentiation capacities. ISCs in the crypt base are sustained by their surrounding niche for precise modulation of self-renewal and differentiation. However, how intestinal cells in the crypt niche and microbiota in enteric cavity regulates ISC stemness remains unclear. Here we show that ISCs are regulated by intestinal nerve cells and macrophage cells in the crypt niche, which are further modulated by microbiota. Enteric serotonergic neurons, along with their secreted neurotransmitter 5-HT, are required for ISC self-renewal. 5-HT activates PGE2 production in macrophages through engagement with its receptors Htr2a/3a, and PGE2 activates Wnt/β-catenin signaling of ISCs via engagement with its receptors Ep1/Ep4. Gut bacterial metabolite valeric acid promotes Tph2 expression through blocking enrichment of NuRD complex onto Tph2 promoter. Our findings reveal the complicated crosstalk between microbiota, intestinal nerve cells, intestinal immune cells and intestinal stem cells, adding a new layer for ISC regulation by niche cells and microbiota.

Extraappendiceal goblet cell carcinoid of the transverse colon

Goblet cell carcinoids (GCC) are a rare subgroup of neuroendocrine tumours which exhibit mixed endocrine and exocrine features and are almost exclusively localised to the appendix. A few cases of extraappendiceal GCC have been reported in the literature however these are exceedingly rare. They are biologically aggressive tumours that are believed to arise from pluripotent intestinal epithelial crypt base stem cells. Given their rarity, no guidelines currently exist for the management of extraappendiceal GCC, however treatment for GCC’s is generally based on their tumour stage in line with staging for typical adenocarcinomas. This case gives an account of a colonic goblet cell carcinoid within the transverse colon encountered in a 66-year-old male. A literature search did not identify any previous documented cases of a GCC at this location.

The Hippo–YAP Signaling as Guardian in the Pool of Intestinal Stem Cells

Despite endogenous insults such as mechanical stress and danger signals derived from the microbiome, the intestine can maintain its homeostatic condition through continuous self-renewal of the crypt–villus axis. This extraordinarily rapid turnover of intestinal epithelium, known to be 3 to 5 days, can be achieved by dynamic regulation of intestinal stem cells (ISCs). The crypt base-located leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) ISCs maintain intestinal integrity in the steady state. Under severe damage leading to the loss of conventional ISCs, quiescent stem cells and even differentiated cells can be reactivated into stem-cell-like cells with multi-potency and contribute to the reconstruction of the intestinal epithelium. This process requires fine-tuning of the various signaling pathways, including the Hippo–YAP system. In this review, we summarize recent advances in understanding the correlation between Hippo–YAP signaling and intestinal homeostasis, repair, and tumorigenesis, focusing specifically on ISC regulation.

The M1 muscarinic acetylcholine receptor in the crypt stem cell compartment mediates intestinal mucosal growth

Maintenance of the highly plastic intestinal epithelium relies upon stem cells localized to intestinal crypts. Recent evidence suggests muscarinic acetylcholine signaling impacts epithelial barrier function, proliferation, and apoptosis. We hypothesized that the intestinal crypt base would express specific muscarinic acetylcholine receptors that drive proliferation in this critical region. Intestinal segments spanning the small bowel were procured from wild-type C57Bl/6 mice to determine muscarinic acetylcholine receptor mRNA expression and create sections on laser capture microdissection slides for analysis of crypt base cells. RT-PCR was performed using primers targeting the five muscarinic acetylcholine receptor subtypes (M1–M5), LGR5, BIII-tubulin, and GAPDH. To determine the effects of muscarinic agonism in vivo, osmotic pumps delivering the M1 muscarinic acetylcholine receptor agonist McN-A-343 were implanted into wild type mice for one week. Segments were harvested, histologic sections created, and morphometric and proliferative parameters measured. In full-thickness intestinal samples, muscarinic acetylcholine receptor subtypes M1–M4 were found in all regions, while M5 was localized to the proximal jejunum. In crypt-base cells, the M1 muscarinic acetylcholine receptor subtype was the only subtype found and was present in all regions. LGR5 was present in all laser capture microdissection samples, indicating the capture of intestinal stem cells. In vivo experiments conducted with McN-A-343 revealed significantly increased villus height, crypt depth, and crypt-cell proliferation. The presence of M1 muscarinic acetylcholine receptor mRNA within the stem cell niche in the intestinal crypt base coupled with increased mucosal growth with M1 receptor stimulation in vivo suggests that the cholinergic system, via the M1 muscarinic acetylcholine receptor, is a critical mediator of intestinal mucosal homeostasis. Impact statement Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

A29 HOPX LABELS A COLONIC STEM CELL THAT CONTRIBUTES TO COLONIC REGENERATION BUT NOT COLONIC TUMORS

Background Colorectal cancer is the 2nd leading cause of cancer death in Canada. In rapidly dividing tissues such as the intestine or colon, only long-lived, multipotent, self-renewing tissue stem cells have longevity to accumulate mutations and serve as the cellular origin of cancer. In the small intestine, genetic fate mapping studies have demonstrated that there are at least two principal stem cell pools: actively cycling, crypt base cells expressing Lgr5, and quiescent cells situated above the crypt base. Clevers and colleagues have previously shown that Lgr5-expressing cells can give rise to cancer upon mutation. Interestingly, when Lgr5+ stem cells are selectively “killed”, intestinal integrity remains intact and other stem cells restore homeostasis. To determine whether another stem cell population can give rise to cancer in the colon, we examined whether the atypical homeobox protein Hopx, marks stem cells in the colon and whether these cells can give rise to colon cancer. Aims In the present study, we aim to determine whether Hopx-expressing cells are colonic stem cells that contribute to gut healing and can give rise to colonic tumours following the loss of APC. Methods To determine whether Hopx expressing cells show stemness, we crossed Hopx-CreERT mice to R26-TdTomato reporter mice. We then conducted genetic lineage tracing studies in the colon during homeostasis and following dextran sodium sulphate (DSS)-induced colitis. To test the function of Hopx expressing cells, Hopx-CreERT mice were also crossed to R26DTR mice and treated with diphtheria toxin (DT) following tamoxifen. These mice were then exposed to either normal drinking water or DSS to determine the role of Hopx+ cells in colonic regeneration. To test whether Hopx expressing cells can serve as a cellular origin for colon cancer, Hopx-CreERT mice were crossed to Apcf/f (floxed) mice. Results Consistent with the labeling of a stem cell, following tamoxifen, Hopx+ cells expressing tdTomato expanded to lineage trace full colonic crypts within 7 days, and labelling was persistent for greater than 6 months. Interestingly, ablation of Hopx+ cells with DT did not alter weight, histological damage or survival during normal homeostasis, however, Hopx+ cell ablation in mice treated with DSS resulted in increased histological damage. Surprisingly, loss of APC in Hopx-expressing cells did not induce colonic adenomas even after 8 months following tamoxifen administration. Conclusions These findings prove that Hopx expressing cells identify a novel colonic stem cell pool that is redundant in homeostasis, but in the context of injury, are essential for epithelial regeneration. Interestingly, Hopx+ cells do not have the capacity to give rise to colorectal adenomas upon loss of the APC gene. Funding Agencies CIHR