Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

Complementation of human papillomavirus type 16 E6 and E7 by Jagged1-specific Notch1-phosphatidylinositol 3-kinase signaling involves pleiotropic oncogenic functions independent of CBF1;Su(H);Lag-1 activation

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.     

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.     

Structural insights into the Notch-modifying glycosyltransferase Fringe

Fringe proteins are beta1,3-N-acetylglucosaminyltransferases that modify Notch receptors, altering their ligand-binding specificity to regulate Notch signaling in development. We present the crystal structure of mouse Manic Fringe bound to UDP and manganese. The structure reveals amino acid residues involved in recognition of donor substrates and catalysis, and a putative binding pocket for acceptor substrates. Mutations of several invariant residues in this pocket impair Fringe activity in vivo.     

Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway

Activated Notch1 (AcN1) alleles cooperate with oncogenes from DNA tumor viruses in transformation of epithelial cells. AcN1 signaling has pleiotropic effects, and suggested oncogenic roles include driving proliferation through cyclin D1 or the generation of resistance to apoptosis on matrix withdrawal through a phosphatidylinositol 3-kinase (PI3K)-PKB/Akt-dependent pathway. Here, we extend the antiapoptotic role for AcN1 by showing inhibition of p53-induced apoptosis and transactivation. Chemical inhibitors of the PI3K pathway block AcN1-induced inhibition of p53-dependent apoptosis and nuclear localization of Hdm2. We show that expression of wild-type p53 does not inhibit synergistic transformation by AcN1 and human papillomavirus E6 and E7 oncogenes. We suggest that activation of Notch signaling may serve as an additional mechanism to inhibit wild-type p53 function in papillomavirus-associated neoplasia.     

Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region

Fringe plays a key role in the specification of boundaries during development by modulating the ability of Notch ligands to activate Notch receptors. Fringe is a fucose-specific beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose moieties on the epidermal growth factor-like (EGF) repeats of Notch. To investigate how the change in sugar structure caused by Fringe modulates Notch activity, we have analyzed the sites of O-fucose and Fringe modification on mouse Notch1. The extracellular domain of Notch1 has 36 tandem EGF repeats, many of which are predicted to be modified with O-fucose. We recently proposed a broadened consensus sequence for O-fucose, C(2)X(3-5)(S/T)C(3) (where C(2) and C(3) represent the second and third conserved cysteines), significantly expanding the potential number of modification sites on Notch. Here we demonstrate that sites predicted using this broader consensus sequence are modified with O-fucose on mouse Notch1, and we present evidence suggesting that the consensus can be further refined to C(2)X(4-5)(S/T)C(3). In particular, we demonstrate that EGF 12, a portion of the ligand-binding site, is modified with O-fucose and that this site is evolutionarily conserved. We also show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all EGF repeats of mouse Notch1. These findings suggest that the Fringes show a preference for O-fucose on some EGF repeats relative to others. This specificity appears to be encoded within the amino acid sequence of the individual EGF repeats. Interestingly, our results reveal that Manic fringe modifies O-fucose both at the ligand-binding site (EGF 12) and in the Abruptex region. These findings provide insight into potential mechanisms by which Fringe action on Notch receptors may influence both the affinity of Notch-ligand binding and cell-autonomous inhibition of Notch signaling by ligand.     

Human papillomavirus type 16 E6 amino acid 83 variants enhance E6-mediated MAPK signaling and differentially regulate tumorigenesis by notch signaling and oncogenic Ras

Oncogenically high-risk human papillomaviruses (HPVs) are causally associated with the progression of major human neoplasia-like cancers of the cervix. Several studies have defined functions of the key E6 and E7 oncoproteins in epithelial cell immortalization. The roles of these oncogenes in the progression of immortalized epithelial cells to invasive tumors are still poorly understood. Here, we establish a novel link between the E6 oncoprotein and activation of mitogen-activated protein kinase (MAPK) signaling and show that this signaling involves Rap1. We find that activated MAPK signaling cooperates with deregulated Notch1 signaling to recreate features of HPV-driven invasive cervical carcinomas. We extend our analysis to evaluate an E6 (amino acid [aa] 83) variant that has been linked to invasive tumors. The variant enhances MAPK signaling and cooperative transformation with deregulated Notch1 signaling. Unlike E6, this variant surprisingly inhibits oncogenic Ras-mediated transformation. Our data reveal that the quantitative differences in activation of MAPK signaling by E6 and its variant correlate with differences in cooperative transformation with other signaling pathways, thus suggesting that thresholds of MAPK activation may define permissive conditions for other signaling pathways in tumorigenesis. Epidemiological studies have suggested the importance of E6 aa 83 variants in invasive carcinomas; our data support a key deterministic role for this variant in human cervical tumorigenesis. These observations, along with our recent data showing that deregulated Notch signaling activates phosphatidylinositol 3-kinase signaling, strengthen the possibility of the existence of Ras-independent mechanisms to recreate signaling through classical Ras effector pathways.     

Angiotensin II contributes to renal fibrosis independently of Notch pathway activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.     

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.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Evidence for the notch signaling pathway on the role of estrogen in angiogenesis

We have investigated the molecular mechanisms involved in 17 beta-estradiol-induced angiogenic pathway. We show here that 17 beta-estradiol promoted a 6-fold increase in Jagged1 expression and an 8-fold increase in Notch1 expression by cDNA arrays in breast cancer MCF7 cells. Interestingly, Jagged1 was abrogated by incubation with the estrogen antagonist, ICI182,780. A similar up-regulation of both Notch1 receptor and Jagged1 ligand was found in endothelial cells. Additionally, imperfect estrogen-responsive elements were found in the 5' untranslated region of Notch1 and Jagged1 genes. Treatment with 17 beta-estradiol also led to an activation of Notch signaling in MCF7 cells expressing Notch1 reporter gene or by promoting Jagged1-induced Notch signaling in coculture assays. Inoculation of MCF7 cells in 17 beta-estradiol-treated nude mice resulted in up-regulation of Notch1 expression as well as increased number of tumor microvessels in comparison to placebo-treated mice. Notch1-expressing endothelial cell cultures formed cord-like structures on Matrigel in contrast to cells expressing a dominant-negative form of Notch1, emphasizing the relevance of Notch1 pathway in vessel assembly. Finally, Notch1-expressing MCF7 cells up-regulated hypoxia-inducible factor 1 alpha gene, a well-known angiogenic factor that clustered with Notch1 gene. This study implicates Notch signaling in the cross talk between 17 beta-estradiol and angiogenesis.     

Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells

Background

Hypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown.

Methodology/Principal Findings

miRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT.

Conclusions/Significance

Our study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.     

An O-fucose site in the ligand binding domain inhibits Notch activation

Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. Here, we describe the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. Our results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicate that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling.     

Lunatic fringe, manic fringe, and radical fringe recognize similar specificity determinants in O-fucosylated epidermal growth factor-like repeats

Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific beta1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.