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.     

Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta‐like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast‐specific manner, we investigated the ligand‐specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐induced Notch signaling was responsible for the expansion of the bone‐forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1‐Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast‐specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast‐osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1‐mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand‐specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Distinct Biological Roles for the Notch Ligands Jagged-1 and Jagged-2

Notch signaling is activated in a subset of non-small cell lung cancer cells because of overexpression of Notch3, but the role of Notch ligands has not been fully defined. On the basis of gene expression profiling of a panel of non-small cell lung cancer cell lines, we found that the predominant Notch ligands were JAG1, JAG2, DLL1, and DLL3. Given that Notch ligands reportedly have overlapping receptor binding specificities, we postulated that they have redundant biological roles. Arguing against this hypothesis, we found that JAG1 and JAG2 were differentially regulated; JAG1 expression was dependent upon epidermal growth factor receptor (EGFR) activation in HCC827 cells, which require EGFR for survival, whereas JAG2 expression was EGFR-independent in these cells. Furthermore, HCC827 cells underwent apoptosis following depletion of JAG1 but not JAG2, whereas co-culture experiments revealed that depletion of JAG2, but not JAG1, enhanced the ability of HCC827 cells to chemoattract THP-1 human monocytes. JAG2-depleted HCC827 cells expressed high levels of inflammation-related genes, including interleukin 1 (IL1) and a broad range of IL1-regulated cytokines, which was attenuated by inhibition of IL1 receptor (IL1R). Our findings suggest that JAG1 and JAG2 have distinct biological roles including a previously undiscovered role for JAG2 in regulating the expression of cytokines that can promote antitumor immunity.     

Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling

NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1-36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling.     

Dicer is required for the maintenance of notch signaling and gliogenic competence during mouse retinal development

MicroRNAs (miRNAs) are 19-25 nucleotide RNAs that regulate messenger RNA translation and stability. Recently, we performed a conditional knockout (CKO) of the miRNA-processing enzyme Dicer during mouse retinal development and showed an essential role for miRNAs in the transition of retinal progenitors from an early to a late competence state (Georgi and Reh [2010]: J Neurosci 30:4048-4061). Notably, Dicer CKO progenitors failed to express Ascl1 and generated ganglion cells beyond their normal competence window. Because Ascl1 regulates multiple Notch signaling components, we hypothesized that Notch signaling is downregulated in Dicer CKO retinas. We show here that Notch signaling is severely reduced in Dicer CKO retinas, but that retinal progenitors still retain a low level of Notch signaling. By increasing Notch signaling in Dicer CKO progenitors through constitutive expression of the Notch intracellular domain (NICD), we show that transgenic rescue of Notch signaling has little effect on the competence of retinal progenitors or the enhanced generation of ganglion cells, suggesting that loss of Notch signaling is not a major determinant of these phenotypes. Nevertheless, transgenic NICD expression restored horizontal cells, suggesting an interaction between miRNAs and Notch signaling in the development of this cell type. Furthermore, while NICD overexpression leads to robust glial induction in control retinas, NICD overexpression was insufficient to drive Dicer-null retinal progenitors to a glial fate. Surprisingly, the presence of transgenic NICD expression did not prevent the differentiation of some types of retinal neurons, suggesting that Notch inactivation is not an absolute requirement for the initial stages of neuronal differentiation.     

Pofut1 is required for the proper localization of the Notch receptor during mouse development

Protein O-fucosyltransferase 1 (Pofut1), which catalyzes the addition of O-linked fucose to the EGF domains of the Notch receptor, is indispensable for Notch signaling activation. However, the mechanism of action of Pofut1 in mice is still unclear. Mouse embryos lacking Pofut1 shows defects in valve formation and trabeculation in the cardiovascular system, which are almost identical abnormalities to those of the RBP-Jk mutants. In our current study, we have examined the epistatic relationship between the functions of Pofut1 and activated-Notch1 (NICD1) by taking advantage of the fact that forced expression of NICD1 results in myocardial defects. These defects were still evident in NICD1-expressing embryos irrespective of the presence or absence of Pofut1, which indicates that Pofut1 is required for Notch signaling upstream of NICD1. We further found that Pofut1-null cells do not possess normally localized Notch1 receptors, which may results in their lack of interaction with the Dll1 ligand in the presomitic mesoderm where Notch signaling plays a pivotal role. We propose that altered trafficking pathways may account for the abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum in Pofut1-null mouse embryos.     

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.     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.     

Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1

The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos.     

O-Fucosylation of DLL3 Is Required for Its Function during Somitogenesis

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.     

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.     

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.     

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

Background

Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.

Methods

We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.

Results

Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.

Conclusions

Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.     

The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear

The mammalian auditory sensory epithelium, the organ of Corti, contains sensory hair cells and nonsensory supporting cells arranged in a highly patterned mosaic. Notch-mediated lateral inhibition is the proposed mechanism for creating this sensory mosaic. Previous work has shown that mice lacking the Notch ligand JAG2 differentiate supernumerary hair cells in the cochlea, consistent with the lateral inhibitory model. However, it was not clear why only relatively modest increases in hair cell production were observed in Jag2 mutant mice. Here, we show that another Notch ligand, DLL1, functions synergistically with JAG2 in regulating hair cell differentiation in the cochlea. We also show by conditional inactivation that these ligands probably signal through the NOTCH1 receptor. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate, rather than through excess proliferation. Although these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the organ of Corti. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.     

Cre-conditional expression of constitutively active Notch1 in transgenic mice

The Notch signaling pathway plays a critical role during mammalian development. To bypass embryonic lethality associated with constitutive Notch1 signaling, we created transgenic mice with a floxed beta-geo/stop signal between a cytomegalo virus promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1). IC-Notch1 is activated upon introduction of Cre recombinase and it is coexpressed with an enhanced green fluorescent protein or human placental alkaline phosphatase reporter. We created three IC-Notch1 transgenic mouse lines and crossed them to a general Cre deletor mouse line, pCX-Cre. The double transgenic IC-Notch1/pCX-Cre embryos have widespread expression of IC-Notch1 and reporters and die before 10.5 days of gestation. Morphological and histological analysis of the double transgenic embryos indicated growth arrest and various developmental defects, including lack of neural tube closure, disorganized somites, and disrupted vasculature. The conditional IC-Notch1 expressing transgenic mice provide a unique tool to investigate the Notch pathway using tissue-specific Cre mice and inducible Cre systems.