Toward a quantitative understanding of the Wnt/beta-catenin pathway through simulation and experiment

Wnt signaling regulates cell survival, proliferation, and differentiation throughout development and is aberrantly regulated in cancer. The pathway is activated when Wnt ligands bind to specific receptors on the cell surface, resulting in the stabilization and nuclear accumulation of the transcriptional co‐activator β‐catenin. Mathematical and computational models have been used to study the spatial and temporal regulation of the Wnt/β‐catenin pathway and to investigate the functional impact of mutations in key components. Such models range in complexity, from time‐dependent, ordinary differential equations that describe the biochemical interactions between key pathway components within a single cell, to complex, multiscale models that incorporate the role of the Wnt/β‐catenin pathway target genes in tissue homeostasis and carcinogenesis. This review aims to summarize recent progress in mathematical modeling of the Wnt pathway and to highlight new biological results that could form the basis for future theoretical investigations designed to increase the utility of theoretical models of Wnt signaling in the biomedical arena

Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-gamma by a novel stem cell niche tracking method

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-gamma (IFN-gamma), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-gamma remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-gamma by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-gamma. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-gamma, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche. (C) 2021 The Authors. Published by Elsevier Inc

Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells

The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites

Expression and Localization of Paneth Cells and Their alpha-Defensins in the Small Intestine of Adult Mouse

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting alpha-defensin. In Institute of Cancer Research (ICR) mice, alpha-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that alpha-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of alpha-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 10(3) to 1 x 10(6) copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their alpha-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity

Paneth cell alpha-defensin misfolding correlates with dysbiosis and ileitis in Crohn's disease model mice

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and alpha-defensin misfolding have been reported in CD patients. Because alpha-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, alpha-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form alpha-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form alpha-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form alpha-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell alpha-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32–128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution