Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-Catenin signaling often intersect in stem and progenitor cells and regulate one another transcriptionally. The biological outcome of signaling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here, we show that membrane-bound Notch physically associates with unphosphorylated (active) β-Catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-Catenin protein. Notch-dependent regulation of β-Catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did, however, require the endocytic adaptor protein, Numb, and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-Catenin protein levels in stem and progenitor cells.

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Notch is an ancient transmembrane receptor with critical roles in cell-fate choices. While the “canonical” Notch pathway and its core members are well established -- involving ligand-induced cleavage of Notch for transcriptional regulation -- it has been unclear whether Notch can also function independent of ligand and transcription (‘non-canonically’) through a common mechanism. Recent studies suggest that Notch can non-canonically exert its biological functions by posttranslationally targeting Wnt/β-catenin signaling, an important cellular and developmental regulator. The non-canonical Notch pathway appears to be highly conserved from flies to mammals. Here, we discuss the emerging conserved mechanism and role of ligand/transcription-independent Notch signaling in cell and developmental biology.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell–mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

Arrhythmogenic cardiomyopathy (ACM) is characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. We previously reported that SB216763 (SB2), annotated as a GSK3β inhibitor, reverses disease phenotypes in a zebrafish model of ACM. Here, we show that SB2 prevents myocyte injury and cardiac dysfunction in vivo in two murine models of ACM at baseline and in response to exercise. SB2-treated mice with desmosome mutations showed improvements in ventricular ectopy and myocardial fibrosis/inflammation as compared with vehicle-treated (Veh-treated) mice. GSK3β inhibition improved left ventricle function and survival in sedentary and exercised Dsg2mut/mut mice compared with Veh-treated Dsg2mut/mut mice and normalized intercalated disc (ID) protein distribution in both mutant mice. GSK3β showed diffuse cytoplasmic localization in control myocytes but ID redistribution in ACM mice. Identical GSK3β redistribution is present in ACM patient myocardium but not in normal hearts or other cardiomyopathies. SB2 reduced total GSK3β protein levels but not phosphorylated Ser 9–GSK3β in ACM mice. Constitutively active GSK3β worsens ACM in mutant mice, while GSK3β shRNA silencing in ACM cardiomyocytes prevents abnormal ID protein distribution. These results highlight a central role for GSKβ in the complex phenotype of ACM and provide further evidence that pharmacologic GSKβ inhibition improves cardiomyopathies due to desmosome mutations.

Introduction: Inflammation is a prominent feature of arrhythmogenic cardiomyopathy (ACM), but whether it contributes to the disease phenotype is not known. To define the role of inflammation in the pathogenesis of ACM, we characterized effects of inhibition of inflammatory signaling in ACM models in vitro and in vivo, and in cardiac myocytes from patient induced pluripotent stem cells (hiPSCs). Results: Activation of NFκB signaling, indicated by increased expression and nuclear accumulation of phospho-RelA/p65, occurs in both an in vitro model of ACM (expression of JUP 2157del2 in neonatal rat ventricular myocytes), and in a robust murine model of ACM (homozygous knock-in of mutant desmoglein-2; Dsg2 mut/mut) that recapitulates the cardiac manifestations seen in ACM patients. Bay 11-7082, a small molecule inhibitor of NFκB signaling, prevented development of ACM disease features in vitro (abnormal redistribution of intercalated disk proteins, myocyte apoptosis, release of inflammatory cytokines) and in vivo (myocardial necrosis and fibrosis, LV contractile dysfunction, ECG abnormalities). Hearts of Dsg2 mut/mut mice expressed markedly increased levels of inflammatory cytokines and chemotactic molecules which were attenuated by Bay 11-7082. Salutary effects of Bay 11-7082 correlated with the extent to which production of selected cytokines had been blocked. NFκB signaling was also activated in cardiac myocytes derived from a patient with ACM. These cells produced and secreted abundant inflammatory cytokines under basal conditions, and this was also greatly reduced by Bay 11-7082. Conclusions: Inflammatory signaling is activated in ACM and it drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM.

The discovery of the first heart field (FHF) and the second heart field (SHF) led us to understand how cardiac lineages and structures arise during development. However, it remains unknown how they are specified. Here, we generate precardiac spheroids with pluripotent stem cells (PSCs) harboring GFP/RFP reporters under the control of FHF/SHF markers, respectively. GFP+ cells and RFP+ cells appear from two distinct areas and develop in a complementary fashion. Transcriptome analysis shows a high degree of similarities with embryonic FHF/SHF cells. Bmp and Wnt are among the most differentially regulated pathways, and gain- and loss-of-function studies reveal that Bmp specifies GFP+ cells and RFP+ cells via the Bmp/Smad pathway and Wnt signaling, respectively. FHF/SHF cells can be isolated without reporters by the surface protein Cxcr4. This study provides novel insights into understanding the specification of two cardiac origins, which can be leveraged for PSC-based modeling of heart field/chamber-specific disease.

Recent remarkable studies have reported that clonogenic putative cardiac stem cells (CSCs) with cardiomyogenic potential migrate from heart tissue biopsies during ex vivo culture, and that these CSCs self-organize into spontaneously beating cardiospheres (CSs). Such data have provided clear promise that injured heart tissue may be repaired by stem cell therapy using autologous CS-derived cells. By further examining CSs from the original CS protocol using immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and microscopic analysis, we here report a more mundane result: that spontaneously beating CSs from neonatal rats likely consist of contaminating myocardial tissue fragments. Thus, filtering away these tissue fragments resulted in CSs without cardiomyogenic potential. Similar data were obtained with CSs derived from neonatal mice as wells as adult rats/mice. Additionally, using in vitro culture, fluorescence-activated cell sorting, and immunofluorescence, we demonstrate that these CSs are generated by cellular aggregation of GATA-4+/collagen I+/α-smooth muscle actin (SMA)+/CD45− cells rather than by clonal cell growth. In contrast, we found that the previously proposed CS-forming cells, dubbed phase bright cells, were GATA-4−/collagen I−/α-SMA−/CD45+ and unable to form CSs by themselves. Phenotypically, the CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential. Our data imply that the murine CS model is unsuitable as a source of CSCs with cardiomyogenic potential, a result that is in contrast to previously published data. We therefore suggest, that human CSs should be further characterized with respect to phenotype and differentiation potential before initiating human trials. Disclosure of potential conflicts of interest is found at the end of this article.