PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo.

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/ß-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/ß-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.

Stromal control of intestinal development and the stem cell niche.

Intestinal homeostasis is dependent on the continuous production of differentiated epithelial cells from a sustainable and resilient stem cell compartment. Wnt/ß-catenin signaling plays a central role in this process, cooperating with R-spondins, growth factors and regulators of the TGF-ß/BMP pathway to generate a specialized tissue microenvironment that regulates the intestinal stem cell niche. Recent studies revealed that many of these factors are produced in a paracrine manner by specialized cell populations that reside in the subepithelial stroma. These stromal signal-producing cells, variously called telocytes and myofibroblasts, can be identified by expression of specific genes including PdgfRa, Gli1 and FoxL1. In this review we discuss how the intestinal stem cell niche is established during development and then sustained during adult intestinal homeostasis by these stromal cell populations. The signaling stroma cells regulate intestinal stem cell development into different epithelial lineages and play an important role in the response to environmental stresses.

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.

Wnt/ß-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth cells as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (Porcn(Del)/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating that epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of Porcn(Del) organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable and that stromal production of Wnts can fully support normal murine intestinal homeostasis.

Gut microbiota accelerate tumor growth via c-jun and STAT3 phosphorylation in APCMin/+ mice.

Chronic inflammation is increasingly recognized as a major contributor of human colorectal cancer (CRC). While gut microbiota can trigger inflammation in the intestinal tract, the precise signaling pathways through which host cells respond to inflammatory bacterial stimulation are unclear. Here, we show that gut microbiota enhances intestinal tumor load in the APC(Min/+) mouse model of CRC. Furthermore, systemic anemia occurs coincident with rapid tumor growth, suggesting a role for intestinal barrier damage and erythropoiesis-stimulating mitogens. Short-term stimulation assays of murine colonic tumor cells reveal that lipopolysaccharide, a microbial cell wall component, can accelerate cell growth via a c-Jun/JNK activation pathway. Colonic tumors are also infiltrated by CD11b+ myeloid cells expressing high levels of phospho-STAT3 (p-Tyr705). Our results implicate the role of gut microbiota, through triggering the c-Jun/JNK and STAT3 signaling pathways in combination with anemia, in the acceleration of tumor growth in APC(Min/+) mice.

Enterococcus faecalis from newborn babies regulate endogenous PPARgamma activity and IL-10 levels in colonic epithelial cells.

The postembryonic development of the gastrointestinal tract is subject to regulation by the colonizing microbiota. This maturation process requires the commensal bacteria to cross-talk with host cells by way of recognizing receptors and inducing signaling pathways to activate transcription factors such as the nuclear receptors. Here, we show that in colonic cell lines and in primary colonic cells, Enterococcus faecalis isolated from newborn babies possess the ability to regulate peroxisome proliferator-activated receptor-gamma1 (PPARgamma1) activity through phosphorylation. This results in elevated DNA binding and transcriptional activation of downstream target genes, including IL-10, a cytokine known to modulate innate immune function. Furthermore, phosphorylation appears tightly regulated as phospho-PPARgamma1 becomes an immediate substrate for degradation possibly to curtail any extended transactivation. The involvement of PPARgamma1 in a myriad of physiological processes further confirms that microflora-driven regulation might be important for a number of homeostatic strategies in the gut.

Vangl2 promotes the formation of long cytonemes to enable distant Wnt/ß-catenin signaling.

Wnt signaling regulates cell proliferation and cell differentiation as well as migration and polarity during development. However, it is still unclear how the Wnt ligand distribution is precisely controlled to fulfil these functions. Here, we show that the planar cell polarity protein Vangl2 regulates the distribution of Wnt by cytonemes. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, Vangl2 activation generates extremely long cytonemes, which branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/ß-catenin signaling in the surrounding cells. Concordantly, Vangl2 inhibition causes fewer and shorter cytonemes to be formed and reduces paracrine Wnt/ß-catenin signaling. A mathematical model simulating these Vangl2 functions on cytonemes in zebrafish gastrulation predicts a shift of the signaling gradient, altered tissue patterning, and a loss of tissue domain sharpness. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we demonstrate that Vangl2 is fundamental to paracrine Wnt/ß-catenin signaling by controlling cytoneme behaviour.

Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells.

Intestinal homeostasis depends on a slowly proliferating stem cell compartment in crypt cells, followed by rapid proliferation of committed progenitor cells in the transit amplifying (TA) compartment. The balance between proliferation and differentiation in intestinal stem cells (ISCs) is regulated by Wnt/ß-catenin signaling, although the mechanism remains unclear. We previously targeted PORCN, an enzyme essential for all Wnt secretion, and demonstrated that stromal production of Wnts was required for intestinal homeostasis. Here, a PORCN inhibitor was used to acutely suppress Wnt signaling. Unexpectedly, the treatment induced an initial burst of proliferation in the stem cell compartment of the small intestine, due to conversion of ISCs into TA cells with a loss of intrinsic ISC self-renewal. This process involved MAPK pathway activation, as the proliferating cells in the base of the intestinal crypt contained phosphorylated ERK1/2, and a MEK inhibitor attenuated the proliferation of ISCs and their differentiation into TA cells. These findings suggest a role for Wnt signaling in suppressing the MAPK pathway at the crypt base to maintain a pool of ISCs. The interaction between Wnt and MAPK pathways in vivo has potential therapeutic applications in cancer and regenerative medicine.

Wnt/PCP controls spreading of Wnt/ß-catenin signals by cytonemes in vertebrates.

Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/ß-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/ß-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.

CEACAM1 is a potent regulator of B cell receptor complex-induced activation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is a member of the immunoglobulin (Ig) superfamily, previously characterized as an adhesion and signaling molecule in epithelial, endothelial, and hematopoietic cells. Here, we show that the CEACAM1 isoform expression pattern is different in nonactivated and activated primary mouse B lymphocytes and that CEACAM1 influences B cell receptor complex-mediated activation. A CEACAM1-specific monoclonal antibody strongly triggered proliferation of mouse B cells when combined with surface IgM cross-linking. However, anti-CEACAM1 was not mitogenic when added alone. The proliferation was more pronounced and lasted longer as compared with other activators of B cells, such as anti-IgM in the presence of interleukin-4 or lipopolysaccharide. A similar, costimulatory effect was exerted by CEACAM1-expressing fibroblasts, indicating that homophilic CEACAM1-CEACAM1 cell-mediated binding is the physiological stimulus for CEACAM1-triggered B cell signaling. The anti-CEACAM1/anti-IgM-activated cells aggregated in a lymphocyte function-associated antigen-1-dependent manner. Furthermore, cells that were activated by anti-CEACAM1/anti-IgM secreted Ig but did not go through Ig class-switching. Anti-CEACAM1 induced phosphorylation of c-Jun N-terminal kinase (stress-activated protein kinase) but did not activate the extracellular signal-regulated kinase or p38 mitogen-activated protein kinases.

SB939, a novel potent and orally active histone deacetylase inhibitor with high tumor exposure and efficacy in mouse models of colorectal cancer.

Although clinical responses in liquid tumors and certain lymphomas have been reported, the clinical efficacy of histone deacetylase inhibitors in solid tumors has been limited. This may be in part due to the poor pharmacokinetic of these drugs, resulting in inadequate tumor concentrations of the drug. SB939 is a new hydroxamic acid based histone deacetylase inhibitor with improved physicochemical, pharmaceutical, and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines. It has very favorable pharmacokinetic properties after oral dosing in mice, with >4-fold increased bioavailability and 3.3-fold increased half-life over suberoylanilide hydroxamic acid (SAHA). In contrast to SAHA, SB939 accumulates in tumor tissue and induces a sustained inhibition of histone acetylation in tumor tissue. These excellent pharmacokinetic properties translated into a dose-dependent antitumor efficacy in a xenograft model of human colorectal cancer (HCT-116), with a tumor growth inhibition of 94% versus 48% for SAHA (both at maximum tolerated dose), and was also effective when given in different intermittent schedules. Furthermore, in APC(min) mice, a genetic mouse model of early-stage colon cancer, SB939 inhibited adenoma formation, hemocult scores, and increased hematocrit values more effectively than 5-fluorouracil. Emerging clinical data from phase I trials in cancer patients indicate that the pharmacokinetic and pharmacologic advantages of SB939 are translated to the clinic. The efficacy of SB939 reported here in two very different models of colorectal cancer warrants further investigation in patients.

The long and winding road to gut homeostasis.

PURPOSE OF REVIEW: The open ecosystem of the alimentary tract, harboring approximately 1 kg of bacteria, exhibits a rapid, but tightly controlled turnover. Impaired nuclear receptor function can give rise to perturbation in the gut, leading to inflammation and possibly neoplasia. Intriguingly, bacteria-dependent signaling pathways can modulate, and in turn be modulated by, a subset of nuclear receptors. This review attempts to highlight how microbes and nuclear receptors could jointly regulate gut homeostasis. RECENT FINDINGS: Commensal bacteria can utilize peroxisome proliferator activated receptor-gamma-dependent nuclear export of RelA as a novel mechanism to attenuate inflammatory signals triggered by a pathogen. Other nuclear receptors, such as liver X receptor, vitamin D receptor and farnesoid X receptor were also recently shown to interact with bacteria-induced mammalian inflammatory pathways. Although details of this interplay are still being unraveled, a role for these and other nuclear receptors in gastrointestinal inflammation and possibly neoplasia is beyond dispute. SUMMARY: The commensal microflora is being accorded due importance in regulating homeostasis of the gastrointestinal tract. Recent data suggest that the molecular messengers used by these bacteria include nuclear receptors. Exploiting mechanisms of nuclear receptor activity as drug targets, together with a detailed knowledge of the microbiota, could improve our understanding of gut-related ailments, and aid in mitigating their symptoms.

The Wnt/beta-catenin signaling pathway targets PPARgamma activity in colon cancer cells.

Control of colon cell fate in adenocarcinomas is disrupted, in part, due to aberrant Wnt/beta-catenin signaling. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has been implicated in the development of colon cancers. In the adenomatous polyposis coli multiple intestinal neoplasia (APCMin) mouse cancer model, PPARgamma expression in the colonic mucosa is markedly altered. In addition, PPARgamma protein levels are elevated, possibly through sequestration by activated beta-catenin in colon cancer cell lines. Induction of the Wnt/beta-catenin pathway by LiCl also elevated PPARgamma levels and induced PPARgamma-dependent reporter and endogenous target genes. Mechanistically, PPARgamma, through interactions with beta-catenin and T cell transcription factor (Tcf)-4, may be a determinant of cell fate and is likely a target of the Wnt pathway in cancer cells.

Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein.

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Microvilli structures on B lymphocytes: inducible functional domains?

Interactive contact between B lymphocytes and T cells is necessary for their expansion during an immune response. It has been shown that B lymphocytes receive signals from T cells, such as IL-4 and cross-linking of CD40, which are crucial for their differentiation. We previously found that these factors induce formation of microvilli on B cells and that this was correlated with increased homotypic adhesion of B lymphocytes. In this study we have investigated if IL-4 induce segregation of proteins to microvilli and lipid rafts. Using immuno-electron microscopy we analyzed cell-surface distribution of molecules involved in B-T cell co-activation. Recruitment to detergent-resistant membrane fractions was analyzed using sucrose gradient centrifugation. We found that microvilli were enriched in ICAM-1 and MHC class II molecules. In contrast, LFA-1 and CD40 were more abundant on the smooth cell surfaces, while B7-2 (CD86) was randomly distributed. We also discovered that depletion of cholesterol, using beta-methyl-cyclodextrin, lowered the number of microvilli, indicating that intact lipid rafts are required for their expression. Moreover, activation of B lymphocytes by lipopolysaccharide (LPS) induced increased expression of GM(1), a marker for lipid rafts. However, although both surface and total levels of GM(1) were similar in B lymphocytes stimulated with either LPS or LPS plus IL-4, GM(1) was mainly expressed on microvilli in LPS plus IL-4-stimulated cells. Taken together, our results indicate that microvilli represent distinct inducible membrane domains that can regulate direct cell-cell interactions via grouping and three-dimensional presentation of cell-surface receptors.