Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway

The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype.     

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.     

Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.     

Cell cycle arrest and apoptosis induced by Notch1 in B cells

Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G(1) phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G(1) cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.     

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.     

Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase

The Notch family of signaling receptors plays key roles in determining cell fate and growth control. Recently, a number of laboratories have shown that O-fucose glycans on the epidermal growth factor (EGF)-like repeats of the Notch extracellular domain modulate Notch signaling. Fringe, a known modifier of Notch function, is an O-fucose specific beta1,3-N-acetylglucosaminyltransferase. The transfer of GlcNAc to O-fucose on Notch by fringe results in the potentiation of signaling by the Delta class of Notch ligands, but causes inhibition of signaling by the Serrate/Jagged class of Notch ligands. Interestingly, addition of a beta1,4 galactose by beta4GalT-1 to the GlcNAc added by fringe is required for Jagged1-induced Notch signaling to be inhibited in a co-culture assay. Thus, both fringe and beta4GalT-1 are modulators of Notch function. Several models have been proposed to explain how alterations in O-fucose glycans result in changes in Notch signaling, and these models are discussed.     

The Notch1/c-Myc Pathway in T Cell Leukemia

The transmembrane receptor Notch1 is a member of the evolutionarily conserved family of developmental regulators originally identified in Drosophila melanogaster. Notch signaling plays essential roles in regulating cell fate in thymic, intestinal, vascular and neuronal development (1-5). Recent studies detect mutations in the Notch1 receptor in roughly half of patients with T cell acute lymphoblastic leukemia (T-ALL) (6). Although expression of an activated Notch1 allele has been shown to cause leukemia in mice, the molecular mechanisms whereby Notch1 mediates cellular transformation are unknown (7). To understand how Notch1 contributes to T cell leukemogenesis, we generated mouse leukemic cell lines where the expression of activated Notch1 was doxycycline-regulated. This cell line was used for gene expression profiling to specifically identify Notch1-regulated genes in leukemia. These studies revealed that Notch1 directly induces the expression of c-myc and that inhibition of Notch1 results in cell cycle arrest and apoptosis and decreased c-myc levels (8). These studies and those performed by Aster, Pear and colleagues in human T-ALL cell lines demonstrate that the direct Notch1-mediated activation of c-myc is required to maintain leukemic growth (8-10). Interestingly, the Notch1/c-Myc oncogenic pathway does not appear limited to T-ALL, as studies by the Efstratiadis group show that expression of intracellular Notch1 leads to mammary tumorigenesis and importantly, transformation appears at least partially c-myc dependent (11). Collectively, these studies begin to delineate how Notch1 mediates cellular transformation and raises the possibility that the Notch1/c-Myc pathway may contribute to human breast cancer and potentially other solid tumors.     

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Age-related differences in insulin-like growth factor-1 receptor signaling regulates Akt/FOXO3a and ERK/Fos pathways in vascular smooth muscle cells

Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin-like growth factor-1 receptor (IGF-1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF-1R signaling changes with age in VSMC is not known. We previously found age-related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the upstream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age-related vascular pathology similar to humans, we compared IGF-1R expression in early passages of VSMC and found a constitutive activation of IGF-1R in VSMC from old compared to young rats, including IGF-1R expression and its tyrosine kinase activity. The link between IGF-1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF-1R with IGF-1 in young cells and attenuation of IGF-1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF-1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip-1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a-targets based on FOXO3a-siRNA treatment. Furthermore, IGF-1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF-1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis.     

Curcumin inhibits proliferation and invasion of osteosarcoma cells through inactivation of Notch-1 signaling

The Notch signaling pathway plays critical roles in human cancers, including osteosarcoma, suggesting that the discovery of specific agents targeting Notch would be extremely valuable for osteosarcoma. Curcumin, a naturally occurring phenolic compound found in curcuma longa, has been shown to inhibit proliferation and induce apoptosis of osteosarcoma cells in vitro and tumor growth in xenotransplant or orthotransplant models. However, the precise molecular mechanisms by which curcumin exerts its antitumor activity remain unclear. Here we used multiple molecular approaches, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the invasion assay, gene transfection, real-time RT-PCR, western blot and gelatin zymography, to investigate whether the downregulation of Notch-1 contributes to curcumin-induced inhibition of proliferation and invasion in osteosarcoma cells. The results showed that curcumin caused marked inhibition of osteosarcoma cell growth and G2/M phase cell cycle arrest. This was associated with concomitant attenuation of Notch-1 and downregulation of its downstream genes, such as matrix metalloproteinases, resulting in the inhibition of osteosarcoma cell invasion through Matrigel. We also found that specific downregulation of Notch-1 via small-interfering RNA prior to curcumin treatment resulted in enhanced inhibition of cell growth and invasion. These results suggest that antitumor activity of curcumin is mediated through a novel mechanism involving inactivation of the Notch-1 signaling pathway. Our data provide the first evidence that the downregulation of Notch-1 by curcumin may be an effective approach for the treatment of osteosarcoma.     

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.     

Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology. Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment. Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells. We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk

Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 receptor (N3ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that are located outside the putative ligand binding domain (LBD) of the receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC, whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling activity. Ligand binding assays with soluble Delta1 and Jagged1 revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. All mutant receptors including Notch3C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa receptor and S1-cleaved receptor fragments. Taken together, these data indicate that CADASIL-associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk, whereas they have similar effects on receptor maturation. Moreover, the data suggest that ligand-induced receptor shedding may not be required for N3ECD deposition in CADASIL.