Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.     

Highly conserved O-fucose sites have distinct effects on Notch1 function

The extracellular domain of mouse Notch1 contains 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified with O-fucose. Previous work from several laboratories has indicated that O-fucosylation plays an important role in ligand mediated Notch activation. Nonetheless, it is not clear whether all, or a subset, of the EGF repeats need to be O-fucosylated. Three O-fucose sites are invariantly conserved in all Notch homologues with 36 EGF repeats (within EGF repeats 12, 26, and 27). To investigate which O-fucose sites on Notch1 are important for ligand-mediated signaling, we mutated the three invariant O-fucose sites in mouse Notch1, along with several less highly conserved sites, and evaluated their ability to transduce Jagged1- and Delta1-mediated signaling in a cell-based assay. Our analysis revealed that mutation of any of the three invariant O-fucose sites resulted in significant changes in both Delta1 and Jagged1 mediated signaling, but mutations in less highly conserved sites had no detectable effect. Interestingly, mutation of each invariant site gave a distinct effect on Notch function. Mutation of the O-fucose site in EGF repeat 12 resulted in loss of Delta1 and Jagged1 signaling, while mutation of the O-fucose site in EGF repeat 26 resulted in hyperactivation of both Delta1 and Jagged1 signaling. Mutation of the O-fucose site in EGF repeat 27 resulted in faulty trafficking of the Notch receptor to the cell surface and a decreased S1 processing of the receptor. These results indicate that the most highly conserved O-fucose sites in Notch1 are important for both processing and ligand-mediated signaling in the context of a cell-based signaling assay.     

Proteome analysis of cultivated vascular smooth muscle cells from a CADASIL patient

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementing disease caused by mutations in the NOTCH3 gene, most which are missense mutations leading to an uneven number of cysteine residues in epidermal growth factor-like repeats in the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on the VSMCs of small and middle-sized arteries. Recent studies have demonstrated that impairment of Notch3 signaling is not the primary cause of the disease. In the present study we used proteomic analysis to characterize the protein expression pattern of a unique material of genetically genuine cultured human CADASIL VSMCs. We identified 11 differentially expressed proteins, which are involved in protein degradation and folding, contraction of VSMCs, and cellular stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic reticulum stress and activation of unfolded protein response, leading to increased reactive oxygen species and inhibition of cell proliferation. In addition, upregulation of contractile proteins suggests an alteration in the signaling system of VSMC contraction. The accumulation of N3ECD on the cell surface possibly upregulates the angiotensin II regulatory feedback loop and thereby enhances the readiness of the cells to respond to angiotensin II stimulation.     

Intrinsic Selectivity of Notch 1 for Delta-like 4 Over Delta-like 1

Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6–15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8–10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6–15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways.     

Notch signaling in normal and disease States: possible therapies related to glycosylation

The Notch signaling pathway is involved in a wide variety of highly conserved developmental processes in mammals. Importantly, mutations of the Notch protein and components of its signaling pathway have been implicated in an array of human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL, Alagille Syndrome, Spondylocostal Dysostosis). In mammals, Notch becomes activated upon binding of its extracellular domain to ligands (Delta and Jagged/Serrate) that are present on the surface of apposed cells. The extracellular domain of Notch contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats. Many of these EGF repeats are modified at evolutionarily-conserved consensus sites by an unusual form of O-glycosylation called O-fucose. Work from several groups indicates that O-fucosylation plays an important role in ligand mediated Notch signaling. Recent evidence also suggests that the enzyme responsible for addition of O-fucose to Notch, protein O-fucosyltransferase-1 (POFUT1), may serve a quality control function in the endoplasmic reticulum. Additionally, some of the O-fucose moieties are further elongated by the action of members of the Fringe family of beta-1,3-N-acetylglucosaminyltransferases. The alteration in O-fucose saccharide structure caused by Fringe modulates the response of Notch to its ligands. Thus, glycosylation serves an important role in regulating Notch activity. This review focuses on the role of glycosylation in the normal functioning of the Notch pathway. As well, potential roles for glycosylation in Notch-related human diseases, and possible roles for therapeutic targeting of POFUT1 and Fringe in Notch-related human diseases, are discussed.     

Notch signaling in human development and disease

Mutations in Notch signaling pathway members cause developmental phenotypes that affect the liver, skeleton, heart, eye, face, kidney, and vasculature. Notch associated disorders include the autosomal dominant, multi-system, Alagille syndrome caused by mutations in both a ligand (Jagged1 (JAG1)) and receptor (NOTCH2) and autosomal recessive spondylocostal dysostosis, caused by mutations in a ligand (Delta-like-3 (DLL3)), as well as several other members of the Notch signaling pathway. Mutations in NOTCH2 have also recently been connected to Hajdu-Cheney syndrome, a dominant disorder causing focal bone destruction, osteoporosis, craniofacial morphology and renal cysts. Mutations in the NOTCH1 receptor are associated with several types of cardiac disease and mutations in NOTCH3 cause the dominant adult onset disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a vascular disorder with onset in the 4th or 5th decades. Studies of these human disorders and their inheritance patterns and types of mutations reveal insights into the mechanisms of Notch signaling.     

Activation dynamics and signaling properties of Notch3 receptor in the developing pulmonary artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.     

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.     

Roles of Pofut1 and O-fucose in mammalian Notch signaling

Mammalian Notch receptors contain 29-36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1-/- ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1-/- cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.     

Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.     

Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels

Mice with targeted mutations in genes required for Notch signal transduction die during embryogenesis, displaying overt signs of hemorrhage due to defects in their vascular development. Surprisingly, directed expression of a constitutively active form of Notch4 within mouse endothelial cells produces a similar vascular embryonic lethality. Moreover, patients with mutations in Notch3 exhibit the cerebral vascular disorder, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These findings underscore the importance of Notch signaling in vascular development; however, they do not identify the specific functional defect. Here, we report that Notch1, Notch3, Notch4, Delta4, Jagged1 and Jagged2 are all expressed in arteries, but are not expressed by veins. These findings identify an aspect of Notch signaling that could contribute to the mechanism by which this pathway modulates vascular morphogenesis.     

Mice carrying a R142C Notch 3 knock-in mutation do not develop a CADASIL-like phenotype

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, MIM 125310) is a genetic vascular dementia disease that is linked to missense mutations, small in-frame deletions, and splice site mutations in the human Notch 3 gene. Here we describe the generation of a mouse knockin model for one of the most prevalent CADASIL mutations, an arginine to cysteine transition at position 141, R141C, which corresponds to mutation R142C in mouse NOTCH 3. CADASIL(R142C) mice show no apparent CADASIL-like phenotype after histological and MRI analysis. The NOTCH 3 (R142C) receptor is processed normally and does not appear to accumulate the ectodomain, which has been observed in CADASIL patients. We discuss possible reasons for the different outcomes of the same germline CADASIL mutation in mice and humans.     

Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling

Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density lipoprotein receptor-related protein-1 (LRP1), a TSP2 receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the receptor-associated protein (an inhibitor of low density lipoprotein receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel, non-cell autonomous function of LRP1 in cell-cell signaling.     

Notch signalling pathway and human diseases

Several homologs of the Drosophila Notch receptor and its ligands, Delta/Serrate, have been cloned in man. Three human disorders including a neoplasia (a T-cell acute lymphoblastic leukemia/lymphoma), a late onset neurological disease (CADASIL) and a developmental disorder (the Alagille syndrome) are associated with mutations in, respectively, the Notch1, Notch3 and Jagged1 genes, pointing out the broad spectrum of Notch activity in humans. We report herein on what has been learned on the role of these human Notch genes and the mechanisms leading from mutations in those genes to the observed phenotypes.     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.