Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1^Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1^Dll4ki/+ mice, the Dll1^Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.     

γ‐Secretase‐Dependent Cleavage Initiates Notch Signaling from the Plasma Membrane

Notch signaling is critical to animal development, and its dysregulation leads to human maladies ranging from birth defects to cancer. Although endocytosis is currently thought to promote signal activation by delivering activated Notch to endosome‐localized γ‐secretase, the data are controversial and the mechanisms that control Notch endocytosis remain poorly defined. Here, we investigated the relationship between Notch internalization and signaling. siRNA‐mediated depletion studies reveal that Notch endocytosis is clathrin‐dependent and requires epsin1, the adaptor protein complex (AP2) and Nedd4. Moreover, we show that epsin1 interaction with Notch is ubiquitin‐dependent. Contrary to the current model, we show that internalization defects lead to elevated γ‐secretase‐mediated Notch processing and downstream signaling. These results indicate that signal activation occurs independently of Notch endocytosis and that γ‐secretase cleaves Notch at the plasma membrane. These observations support a model where endocytosis serves to downregulate Notch in signal‐receiving cells.     

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.     

The Integrin‐Mediated ILK‐Parvin‐αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells

Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1‐integrin (β1‐itg) to establish apico‐basal polarity and to differentiate in response to prolactin. Downstream of β1‐itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico‐basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue‐specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA‐mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin‐binding guanine nucleotide exchange factor αPix prevented prolactin‐induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation‐specific bifurcation point in β1‐itg‐ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin‐ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408–2417, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

MicroRNA‐760 Inhibits Doxorubicin Resistance in Hepatocellular Carcinoma through Regulating Notch1/Hes1‐PTEN/Akt Signaling Pathway

Accumulating studies have suggested that microRNA‐760 (miR‐760) plays an important role in chemoresistance of various cancer cells. However, whether miR‐760 regulates the chemoresistance of hepatocellular carcinoma (HCC) remains unclear. In this study, we found that miR‐760 was decreased in HCC cell lines, and doxorubicin (Dox) treatment significantly decreased miR‐760 expression in HCC cells. Overexpression of miR‐760 sensitized HCC cells to Dox‐induced cytotoxicity and apoptosis, whereas miR‐760 inhibition showed the opposite effects. Notch1 was predicted as a target gene of miR‐760. miR‐760 negatively regulated Notch1 expression and Notch1/Hes1 signaling. Overexpression of miR‐760 increased PTEN expression and decreased the phosphorylation of Akt. Activation of Notch signaling significantly reversed the inhibitory effect of miR‐760 on Dox‐resistance and abrogated the effect of miR‐760 on the PTEN/Akt signaling pathway in HCC cells. Overall, our results demonstrate that miR‐760 inhibits Dox‐resistance in HCC cells through inhibiting Notch1 and promoting PTEN expression.     

Cyclosporin A Disrupts Notch Signaling and Vascular Lumen Maintenance

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.     

hnRNP I Inhibits Notch Signaling and Regulates Intestinal Epithelial Homeostasis in the Zebrafish

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.