Biomedical subjects

Gerry Weinmaster

Publications and source records attributed to Gerry Weinmaster.

15 recordsLinked to original sources

PubMed2006

Microfibrillar proteins MAGP-1 and MAGP-2 induce Notch1 extracellular domain dissociation and receptor activation.

Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia. Here we show that MAGP-2, a protein present on microfibrils, can also interact with the EGF-like repeats of Notch1. Co-expression of MAGP-2 with Notch1 leads to both cell surface release of the Notch1 extracellular domain and subsequent activation of Notch signaling. Moreover, we demonstrate that the C-terminal domain of MAGP-2 is required for binding and activation of Notch1. Based on the high level of homology, we predicted and further showed that MAGP-1 can also bind to Notch1, cause the release of the extracellular domain, and activate signaling. Notch1 extracellular domain release induced by MAGP-2 is dependent on formation of the Notch1 heterodimer by a furin-like cleavage, but does not require the subsequent ADAM metalloprotease cleavage necessary for production of the Notch signaling fragment. Together these results demonstrate for the first time that the microfibrillar proteins MAGP-1 and MAGP-2 can function outside of their role in elastic fibers to activate a cellular signaling pathway.

Animals↗

PubMed2006

A garden of Notch-ly delights.

Over the past decade, the Notch signaling pathway has been shown to be crucially important for normal metazoan development and to be associated with several human inherited and late onset diseases. The realization that altered Notch signaling contributes at various levels to human disease lead in May to the first meeting dedicated solely to Notch signaling in vertebrate development and disease in Madrid, Spain. Hosted by the Cantoblanco Workshops on Biology and organized by Tom Gridley, José Luis de la Pompa and Juan Carlos Izpisúa Belmonte, the meeting covered diverse aspects of this important signaling pathway.

Animals↗

PubMed2005

The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands.

Animals↗

PubMed2005

The extracellular matrix protein MAGP-2 interacts with Jagged1 and induces its shedding from the cell surface.

Elastic fibers are composed of the protein elastin and a network of 10-12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing full-length Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.

ADAM Proteins↗

PubMed2004

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.

Alkaline Phosphatase↗

PubMed2003

Sequential signaling through Notch1 and erbB receptors mediates radial glia differentiation.

Radial glia cells both generate neurons and physically guide nascent neurons to their target destination in the cortex, and as such they are essential for CNS development. It has been proposed that in the developing cerebellum, neuronal contact induces radial glia formation, however, the mechanisms involved in this process are not well understood. Here we demonstrate that neuronal induction of radial glia formation is the result of sequential signaling through Notch1 and erbB receptors. First, Notch1 activation by neuronal contact induces the glial expression of the brain lipid binding protein (BLBP) and erbB2 genes. Interestingly, two different signaling pathways mediate these effects of Notch1 on transcription, BLBP expression being dependent on Su(H), whereas erbB2 is regulated by a yet unidentified Notch1 pathway. The subsequent increase in erbB2 receptor expression makes the glia more responsive to neuronal NRG, which then induces the morphological transformation into radial glia. Thus, these results unveil some of the mechanisms underlying radial glia formation.

Animals↗

PubMed2003

Basal expression of IkappaBalpha is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and its activator Notch1.

By using the hepatic stellate cell (HSC) as a paradigm for cells that undergo long term re-programming of NF-kappaB-dependent transcription, we have determined a novel mechanism by which mammalian cells establish their basal NF-kappaB activity. Elevation of NF-kappaB activity during HSC activation is accompanied by induction of CBF1 expression and DNA binding activity. We show that the transcriptional repressor CBF1 interacts with a dual NF-kappaB/CBF1-binding site (kappaB2) in the IkappaBalpha promoter. Nucleotide substitutions that disrupt CBF1 binding to the kappaB2 site result in an elevation of IkappaBalpha promoter activity and loss of responsiveness of the promoter to a transfected CBF1 reporter vector. Overexpression of CBF1 in COS1 cells was associated with markedly reduced IkappaBalpha protein expression and elevated NF-kappaB DNA binding activity. CBF1-induced repression of IkappaBalpha promoter activity was reversed in HSC transfected with the Notch1 intracellular domain (NICD). The ability of NICD to enhance IkappaBalpha gene transcription was confirmed in COS1 cells and was found to be dependent on an intact RAM domain of NICD that has been shown previously to help mediate the interaction of NICD with CBF1. One of the mechanisms by which NICD is thought to convert CBF1 into an activator of transcription is via the recruitment of transcriptional co-activators/histone acetylases to gene promoters. Co-transfection of HSC with NICD and p53 caused a diminution of IkappaBalpha promoter activity, by contrast overexpression of p300 enhanced IkappaBalpha promoter function. Taken together, these data suggest that basal IkappaBalpha expression (and as a consequence NF-kappaB activity) is under the control of the various components of the CBF1/Notch signal transduction pathway.

Animals↗

PubMed2003

Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells.

Glycoprotein130 (gp130) and Notch signaling are thought to participate in neural stem cell (NSC) self-renewal. We asked whether gp130 regulates Notch activity in forebrain epidermal growth factor (EGF)-responsive NSCs. Disruption of Notch1 using antisense or a gamma-secretase inhibitor demonstrated a requirement for Notch1 in the maintenance and proliferation of NSCs. Ciliary neurotrophic factor (CNTF) activation of gp130 in NSCs rapidly increased Notch1 expression. NOTCH1 activation, indicated by tumor necrosis factor alpha-converting enzyme (TACE)- and presenilin-mediated processing, also increased. Infusion of EGF+CNTF into adult forebrain lateral ventricles increased periventricular NOTCH1 compared with EGF alone. Neither Hes1 (hairy and enhancer of split) nor Hes5 appeared to mediate gp130-enhanced NOTCH1 signaling that regulates NSC maintenance. This is the first example of a link between gp130 signaling and NOTCH1 in regulating NSC self-renewal.

Amyloid Precursor Protein Secretases↗

PubMed2003

Modulation of notch signaling during somitogenesis.

The Notch signaling pathway is known to govern various aspects of tissue differentiation during embryonic development by mediating local cell-cell interactions that often control cell fate. The conserved components that underlie Notch signaling have been isolated in vertebrates, leading to a biochemical delineation of a core Notch signaling pathway and functional studies of this pathway during embryogenesis. Herein we highlight recent progress in determining how Notch signaling contributes to the development of the vertebrate embryo. We first discuss the role of Notch in the process of segmentation where rapid changes have been shown to occur in both the spatial and temporal aspects of Notch signaling, which are critical for segmental patterning. Indeed, the role of Notch in segmentation re-emphasizes a recurring question in Notch biology: how are the components involved in Notch signaling regulated to ensure their dynamic properties? Second, we address this question by discussing recent work on the biochemical mechanisms that potentially regulate Notch signaling during segmentation, including those that act on the receptors, ligands, and signal transduction apparatus.

Animals↗

PubMed2002

Notch signaling promotes astrogliogenesis via direct CSL-mediated glial gene activation.

In the developing central nervous system (CNS), Notch signaling preserves progenitor pools and inhibits neurogenesis and oligodendroglial differentiation. It has recently been postulated that Notch instructively drives astrocyte differentiation. Whether the role of Notch signaling in promoting astroglial differentiation is permissive or instructive has been debated. We report here that the astrogliogenic role of Notch is in part mediated by direct binding of the Notch intracellular domain to the CSL DNA binding protein, forming a transcriptional activation complex onto the astrocyte marker gene, glial fibrillary acidic protein (GFAP). In addition, we found that, in CSL-/- neural stem cell cultures, astrocyte differentiation was delayed but continued at a normal rate once initiated, suggesting that CSL is involved in regulating the onset of astrogliogenesis. Importantly, although the classical CSL-dependent Notch signaling pathway is intact and able to activate the Notch canonical target promoter during the neurogenic phase, it is unable to activate the GFAP promoter during neurogenesis. Therefore, the effect of Notch signaling on target genes is influenced by cellular context in regulation of neurogenesis and gliogenesis.

Animals↗

PubMed2002

A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling.

Signaling induced through interactions between DSL (Delta, serrate, LAG-2) ligand-signaling cells and Notch-responding cells influences the developmental fate of a wide variety of invertebrate and vertebrate cell types. Consistently with a requirement for direct cell-cell interactions, secreted DSL ligands expressed in flies do not appear to activate Notch signaling but rather produce phenotypes reminiscent of losses in Notch signaling. In contrast, secreted DSL ligands expressed in Caenorhabditis elegans or supplied to mammalian cells in culture produce effects indicative of Notch activation. In fact, engineered secreted DSL ligands have been used to study Notch signaling in neurogenesis, gliogenesis, hematopoeisis, neurite morphogenesis and ligand-induced nuclear translocation of the Notch intracellular domain. Using a recombinant, secreted form of the DSL ligand Delta1, we found that antibody-induced oligomerization (termed "clustering") was required for this soluble ligand to bind specifically to Notch1-expressing cells, undergo internalization, and activate downstream signaling. Interestingly, clustering with either limiting or excess antibody led to ligand binding in the absence of Notch signaling, indicating that ligand binding is necessary but not sufficient for activation of Notch signaling. Moreover, such antibody clustering conditions blocked Notch1 signaling induced by membrane-bound DSL ligands. We propose that multimerization influences whether ligand binding to Notch results in activation or inhibition of downstream signaling and suggest that differences in ligand presentation might account for why secreted forms of DSL ligands have been reported to function as agonists and antagonists of Notch signal transduction.

Animals↗

PubMed2002

Developmental changes in Notch1 and numb expression mediated by local cell-cell interactions underlie progressively increasing delta sensitivity in neural crest stem cells.

Neural stem cells become progressively less neurogenic and more gliogenic with development. Here, we show that between E10.5 and E14.5, neural crest stem cells (NCSCs) become increasingly sensitive to the Notch ligand Delta-Fc, a progliogenic and anti-neurogenic signal. This transition is correlated with a 20- to 30-fold increase in the relative ratio of expression of Notch and Numb (a putative inhibitor of Notch signaling). Misexpression experiments suggest that these changes contribute causally to increased Delta sensitivity. Moreover, such changes can occur in NCSCs cultured at clonal density in the absence of other cell types. However, they require local cell-cell interactions within developing clones. Delta-Fc mimics the effect of such cell-cell interactions to increase Notch and decrease Numb expression in isolated NCSCs. Thus, Delta-mediated feedback interactions between NCSCs, coupled with positive feedback control of Notch sensitivity within individual cells, may underlie developmental changes in the ligand-sensitivity of these cells.

Animals↗

PubMed2002

CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor-like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.

Amino Acid Motifs↗

PubMed2002

Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon.

Notch signaling has a central role in cell fate specification and differentiation. We provide evidence that the Mash1 (bHLH) and Dlx1 and Dlx2 (homeobox) transcription factors have complementary roles in regulating Notch signaling, which in turn mediates the temporal control of subcortical telencephalic neurogenesis in mice. We defined progressively more mature subcortical progenitors (P1, P2 and P3) through their combinatorial expression of MASH1 and DLX2, as well as the expression of proliferative and postmitotic cell markers at E10.5-E11.5. In the absence of Mash1, Notch signaling is greatly reduced and 'early' VZ progenitors (P1 and P2) precociously acquire SVZ progenitor (P3) properties. Comparing the molecular phenotypes of the delta-like 1 and Mash1 mutants, suggests that Mash1 regulates early neurogenesis through Notch-and Delta-dependent and -independent mechanisms. While Mash1 is required for early neurogenesis (E10.5), Dlx1 and Dlx2 are required to downregulate Notch signaling during specification and differentiation steps of 'late' progenitors (P3). We suggest that alternate cell fate choices in the developing telencephalon are controlled by coordinated functions of bHLH and homeobox transcription factors through their differential affects on Notch signaling.

Animals↗

PubMedDate not supplied

Extrinsic and intrinsic factors governing cell fate in cortical progenitor cultures.

Central nervous system germinal zones contain stem cells that generate both neurons and glia. In the recent past, these cells have been isolated, maintained in a variety of culture systems and used in vitro for subsequent characterization of molecular mechanisms underlying brain development. Factors that govern cell fate choices of these neural stem cells have not been fully elucidated, but recent studies suggest that age at the time of culture is an important intrinsic mechanism. Stem cell mitogens and Notch-DSL signaling are significant extrinsic factors. In the current study, we compare neurosphere cultures propagated from animals on embryonic day 12, embryonic day 18 and the day of birth and stimulated to divide by either basic fibroblast growth factor (bFGF) or transforming growth factor-alpha (TGF-alpha). As described for other systems, when bFGF was used, clonal neurospheres derived from the youngest age gave rise to a greater percentage of neurons. When TGF-alpha, acting via the epidermal growth factor receptor, was used, this effect was not observed, with neurospheres from younger animals giving rise to a similar percentage of neurons as those derived from older animals suggesting that this growth factor was either stimulating a different population of stem cells to proliferate, or that it was capable of overriding intrinsic mechanisms. Other differences were also observed when the two growth factors were compared, including age-dependent differences in the numbers of putative astrocytes and oligodendrocytes formed. We further assessed age-dependent influences on cell fate by assessing the effects of a lentivirally transduced constitutively activated Notch receptor on cell fate. At all ages studied, Notch activation resulted in a significantly greater number of GFAP-positive cells, seemingly overriding the greater neurogenic potential of younger stem cells. These data suggest that both extrinsic and intrinsic factors differentially regulate cell fate choices of progenitors during cortical development.

Aging↗