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• W2020682158 abstract "Notch1 and β1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and β1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that β1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the β1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of β1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate β1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches. Notch1 and β1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and β1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that β1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the β1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of β1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate β1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches. Integrins and extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; CNS, central nervous system; GF, growth factor(s); ES, embryonic stem; NSC, neural stem cell(s); FGF, fibroblast growth factor; EGF, extracellular growth factor; EGFR, EGF receptor; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione S-transferase; NICD, Notch intracellular domain; VZ, ventricular zone; RGC, radial glial cell; MAPK, mitogen-activated protein kinase. 2The abbreviations used are: ECM, extracellular matrix; CNS, central nervous system; GF, growth factor(s); ES, embryonic stem; NSC, neural stem cell(s); FGF, fibroblast growth factor; EGF, extracellular growth factor; EGFR, EGF receptor; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione S-transferase; NICD, Notch intracellular domain; VZ, ventricular zone; RGC, radial glial cell; MAPK, mitogen-activated protein kinase. molecules play crucial roles during embryogenesis (2De Arcangelis A. Georges-Labouesse E. Trends Genet. 2000; 16: 389-395Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) in mesoderm development, epithelial morphogenesis, neural tube closure, anchorage to the ECM basal lamina, and central nervous system (CNS) development. In particular, β1-integrins (α6β1) are highly expressed in embryonic stem (ES) cells (3Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. Science. 2002; 298: 597-600Crossref PubMed Scopus (1419) Google Scholar) and in neural stem cells (NSC) (4Campos L.S. Leone D.P. Relvas J.B. Brakebusch C. Fässler R. Suter U ffrench-Constant C. Development. 2004; 131: 3433-3444Crossref PubMed Scopus (267) Google Scholar) and are required for cortical development (5Georges-Labouesse E. Mark M. Messaddeq N. Gansmuller A. Curr. Biol. 1998; 8: 983-986Abstract Full Text Full Text PDF PubMed Google Scholar).Notch1 is a cell surface protein involved in the control of cell fate choices in the developing CNS (1Lewis J. Semin. Cell Dev. Biol. 1998; 9: 583-589Crossref PubMed Scopus (349) Google Scholar). This transmembrane receptor is involved in stem cell maintenance (6Hitoshi S. Alexson T. Tropepe V. Donoviel D. Elia A.J. Nye J.S. Conlon R.A. Mak T.W. Bernstein A. van der Kooy D. Genes Dev. 2002; 16: 846-858Crossref PubMed Scopus (548) Google Scholar) and promotes glial and neural fates in a stepwise manner, first by inhibiting neuronal fate and promoting glial fate and second by promoting astrocyte differentiation (7Grandbarbe L. Bouissac J. Rand M. Hrabe de Angelis M. Artavanis-Tsakonas S. Mohier E. Development. 2003; 130: 1391-1402Crossref PubMed Scopus (214) Google Scholar). Notch1 also plays a role in the control of neurite extension in mammalian cells and in axon growth in Drosophila (8Sestan N. Artavanis-Tsakonas S. Rakic P. Science. 1999; 286: 741-746Crossref PubMed Scopus (494) Google Scholar, 9Franklin J.L. Berechid B.E. Cutting F.B. Presente A. Chambers C.B. Foltz D.R. Ferreira A. Nye J.S. Curr. Biol. 1999; 9: 1448-1457Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 10Giniger E. Neuron. 1998; 20: 667-681Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Interestingly, in the immune system Notch1 serves two biologically contrasting functions; it is responsible for the apoptotic cell death of B lymphocytes (11Morimura T. Goitsuka R. Zhang Y. Saito I. Reth M. Kitamura D. J. Biol. Chem. 2000; 275: 36523-36531Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), whereas it promotes the survival of T cells (12Sade H. Krishna S. Sarin A. J. Biol. Chem. 2003; 279: 2937-2944Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The diverse effects of Notch1 activation observed in multiple cell types and at different stages of development suggest the presence of context-dependent control mechanisms. Growth factors (GF) and ECM molecules (acting through integrins) belong to the complex environment that surrounds NSC during development (13Campos L.S. BioEssays. 2005; 27: 698-707Crossref PubMed Scopus (94) Google Scholar) and that affect Notch signaling. For example, FGF-1 and -2 inhibit neural differentiation by affecting (directly or indirectly) the Notch pathway (14Faux C.H. Turnley A.M. Epa R. Cappai R. Bartlett P.F. J. Neurosci. 2001; 21: 5587-5596Crossref PubMed Google Scholar), and EGFR activation leads to Notch signaling during pancreas tumorigenesis (15Miyamoto Y. Maitra A. Ghosh B. Zechner U. Argani P. Iacobuzzo-Donahue C.A. Sriuranpong V. Iso T. Mezoely I.M. Wolfe M.S. Hruban R.H. Ball D.W. Schmid R.M. Leach S.D. Cancer Cell. 2003; 3: 565-576Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Integrins may also be involved in the Notch response during angiogenesis, when Notch4-expressing endothelial cells display β1-integrin in an active, high affinity conformation (16Leong K.G. Hu X. Li L. Noseda M. Larrivee B. Hull C. Hood L. Wong F. Karsan A. Mol. Cell. Biol. 2002; 22: 2830-2841Crossref PubMed Scopus (151) Google Scholar). Furthermore, in zebrafish the boundary cells between developing somites behave differently depending on the levels of Notch activation, and it has been suggested that the extracellular matrix (which differs at the rhombomere boundaries) plays a role in this process (17Blair S.S. Curr. Biol. 2004; 14: R570-R572Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Nevertheless, the coordination between β1-integrin, Notch1, and GF pathways is poorly understood.Lipid rafts are special membrane regions that affect signaling by sorting proteins and lipids into specific membrane domains, where privileged interactions occur. Caveolae are specialized lipid rafts that contain cholesterol, sphingolipids, and caveolins (22-24-kDa membrane proteins, required for the formation of the caveolae) and that serve as scaffolds for signaling molecules.In this paper, we explore how some of the receptors for ECM and GF (that are present on the surface of the NSC) act together with the Notch1 pathway to control the NSC responses to changes in the microenvironment. We discuss the possibility that lipid rafts may play important roles in directing the changes in signaling and the responses to environmental changes that occur during cortical development and may act as integrators of parallel and simultaneous signals originated from integrins, growth factors, and Notch receptors. We conclude that the GF and ECM composition of biological neural stem cell “niches” may affect NSC maintenance and differentiation by affecting Notch signaling, in a context- and time-dependent manner.EXPERIMENTAL PROCEDURESReagents and Antibodies—FGF-2 was obtained from PeProtech, EGF was from Calbiochem, and B27 supplement was from Invitrogen. Antibodies for immunoprecipitation-blocking experiments and Western blots were obtained from Chemicon and Pharmingen (anti-β1 integrins), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (EGFR), Upstate Biotechnology, Inc. (Lake Placid, NY) (EGFR), and Cell Signaling Technology (phosphorylated MAPK and MAPK). Antibodies used for immunohistochemistry included polyclonal Notch1 and caveolin 1 (Santa Cruz Biotechnology), monoclonal anti-Nestin (Pharmingen), and monoclonal anti-β III tubulin (Sigma). All fluorescent secondary antibodies were obtained from Jackson Immunochemicals or Molecular Probes, Inc. (Eugene, OR). The EGF receptor inhibitor AG1478, (Calbiochem) was used at 20 μm. Mixtures of protease and phosphatase inhibitors were from Calbiochem. The remaining products were from Sigma if not otherwise specified.Primary Neurosphere Culture and Preparation of ES Cell-derived NSC—Primary cultures were prepared from newborn and embryonic day 14.5 C57BL/6 mice (postnatal day P0-P2), as previously described (18Jacques T.S. Relvas J.B. Nishimura S. Pytela R. Edwards G.M. Streuli C. ffrench-Constant C. Development. 1998; 125: 3167-3177Crossref PubMed Google Scholar). Briefly, spheres of neural precursors were grown in EGF or FGF-2 (20 ng/ml) from dissociated postnatal day 0-2 mouse forebrain in Dulbecco’s modified Eagle’s medium/Ham’s F-12 supplemented with B27 (19Reynolds B.A. Weiss S. Science. 1992; 255: 1707-1710Crossref PubMed Scopus (4508) Google Scholar, 20Svendsen C.N. Fawcett J.W. Bentlage C. Dunnett S.B. Exp. Brain Res. 1995; 102: 407-414Crossref PubMed Scopus (115) Google Scholar). The culture medium was changed every 3 days.To prepare ES cell-derived NSC, we used a similar approach to the one developed by Bibel et al. (21Bibel M. Richter J. Schrenk K. Tucker K.L. Staiger V. Nat. Neurosci. 2004; 7: 1003-1009Crossref PubMed Scopus (367) Google Scholar). Briefly, we generated embryoid bodies that were exposed to retinoic acid (RA) for 4 days (as described by Bain et al. (22Bain G. Kitchens D. Yao M. Huettner J.E. Gottlieb D.I. Dev. Biol. 1995; 168: 342-357Crossref PubMed Scopus (996) Google Scholar)). Taking into account that a switch in growth factor requirements (from FGF-2 to EGF) occurs in vivo during midneurogenesis (23Lillien L. Raphael H. Development. 2000; 127: 4993-5005Crossref PubMed Google Scholar, 24Martens D.J. Tropepe V. van Der Kooy D. J. Neurosci. 2000; 20: 1085-1095Crossref PubMed Google Scholar), we sequentially exposed the RA-primed embryoid bodies to FGF-2 first and to a mix of FGF-2 and EGF second, in order to simulate the changes in the GF microenvironment that occur during embryonic CNS development.Secondary Neurosphere Formation Assays—Intact primary neurospheres maintained 8-10 days in vitro were mechanically dissociated, and the same number of cells for each condition was plated at low density (5000 cells in 1.5 ml, <5 cells/μl) and grown for 10 days in 20 ng/ml EGF and FGF-2. The number of secondary neurospheres formed was counted, and statistically significant differences between groups were calculated using Student’s t tests.Secondary Neurosphere Formation Assays after Morpholino Treatment or EGFR Inhibition—Intact neurospheres (8-10 days in vitro) were exposed to β1 antisense morpholinos obtained from GeneTools, following the manufacturer’s protocols (available on the World Wide Web at www.gene-tools.com). Control groups were exposed to a missense morpholino with a random sequence. The spheres were then mechanically dissociated, and the same number of cells for each condition were plated at low density (3-4 cells/μl) and grown for 10 days in different growth factor concentrations (20 or 2 ng/ml concentration of either EGF or FGF-2), as described above. The number of secondary neurospheres formed was then counted, and statistically significant differences between groups were calculated using Student’s t tests. Secondary neurosphere formation assays were also done in the presence of an EGFR inhibitor (AG1478, 20 μm), and these experiments were analyzed as described above.Immunohistochemistry—Neurospheres, ES cell-derived NSC, and neonatal or embryonic brain tissue were fixed in 2-4% paraformaldehyde in phosphate-buffered saline. Tissue samples were cryoprotected in 25% sucrose and sectioned (14 μm) prior to immunohistochemistry, except for cell monolayers. The samples were blocked in phosphate-buffered saline (0.1% Triton X-100) containing normal blocking serum and incubated overnight with the appropriate antibodies at 4 °C, followed by incubation with the secondary antibodies and counterstaining with DAPI or sytox green. Pictures were acquired using a Zeiss Axioplan 2 fluorescence microscope and Smartcapture 2 software.GST-NICD Construct Preparation—Briefly, the Notch intracellular domain (NICD) fragment was subcloned from the plasmid pbabe-NICD (kind gift from G. Weinmaster) into the pGEX expression vector to prepare a GST-NICD fusion protein. The fusion protein was produced in Escherichia coli, linked to agarose beads (Amersham Biosciences), and purified using standard protocols. Pull-down assays were performed by incubating total lysates obtained from neurospheres with the GST-NICD or GST-alone beads, for 2 h at room temperature. The beads were then collected, washed, and boiled, and the resulting supernatant was analyzed by SDS-PAGE and immunoblotting as follows.Western Blots and Immunoprecipitations—For Western blotting, neurospheres or ES cell-derived NSC were lysed (10 mm Tris-HCl, 5 mm EDTA, 150 mm NaCl buffer, 1% Triton X-100) in the presence of protease and phosphatase inhibitors (5 μg/ml leupeptin, 2 μg/ml aprotinin, 2 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 2 mm sodium fluoride, 2 mm sodium vanadate, all from Sigma) or the equivalent Calbiochem mixtures. The supernatant was clarified by centrifugation at 14,000 rpm for 20 min at 4 °C. Protein concentrations were determined with a Bio-Rad protein assay with bovine serum albumin as a standard, and equal amounts of protein were loaded in each well. Proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes (Hybond-C; Amersham Biosciences). Membranes were blocked in 10% nonfat dry milk in Tris-buffered saline for 1 h at room temperature. Blots were then incubated with the primary antibodies overnight at 4 °C in milk/Tris-buffered saline containing 0.1% Tween 20 (TBS-T), followed by a 2-h incubation with the appropriate secondary peroxidase-conjugated antibody (Amersham Biosciences). Blots were developed using ECL reagents (Amersham Biosciences), following the manufacturer’s instructions (Amersham Biosciences). For immunoprecipitations, the samples were lysed as previously described. To remove nonspecifically binding proteins, 150-200 μg of proteins were precleared by mixing with agarose beads (A/G plus; Santa Cruz) for 30 min at 4 °C. The samples were then incubated with the adequate antibody in the presence of fresh agarose A/G beads, either at 4 °C overnight or at room temperature for 2 h on a rotating platform. The beads were then washed and boiled for 10 min in Laemmli loading buffer. Equal amounts (as measured by protein assay) were loaded on 10% SDS-polyacrylamide gels and processed for immunoblotting as previously described.Raft Isolation—Isolation of rafts was performed using sucrose gradients, as previously described (25Baron W. Decker L. Colognato H. French-Constant C. Curr. Biol. 2003; 13: 151-155Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Briefly, neurospheres were placed on ice and suspended for 30 min in 0.2 ml of extraction buffer: 50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, and a mixture of protease and phosphatase inhibitors (Calbiochem). Cell lysates were then adjusted to 40% OptiPrep and overlaid with solutions of 30 and 10% Optiprep in the extraction buffer. These gradients were centrifuged for 16 h at 35,000 rpm at 4 °C in an SW50.i rotor (Beckman Instruments). Fractions of equal volume, including the raft (floating fraction) and nonraft (bottom fraction) fractions, were collected and analyzed by SDS-PAGE (10%), followed by immunoblotting. Protein assays were performed on all fractions before immunoblotting to ensure equal loading.Reverse Transcription-PCR—RNA was extracted using the RNAeasy kit (Qiagen), and 0.1 μg of RNA from each sample was used to generate cDNA using the transcriptor first strand cDNA synthesis kit (Roche Applied Science). Reverse transcription-PCR was done using the published primers for Hes5 and glyceraldehyde-3-phosphate dehydrogenase and following the conditions described by Zine et al. (26Zine A. Aubert A. Qiu J. Therianos S. Guillemot F. Kageyama R. de Ribaupierre F. J. Neurosci. 2001; 21: 4712-4720Crossref PubMed Google Scholar)RESULTSβ1-Integrins and Notch1 Co-localize in the Ventricular Zone, in Neurospheres, and in ES Cell-derived NSC—Stem cells from the skin and prostatic epithelia can be identified and isolated by their high β1-integrin expression levels (27Watt F.M. J. Dermatol. Sci. 2002; 28: 173-180Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Collins A.T. Habib F.K. Maitland N.J. Neal D.E. J. Cell Sci. 2001; 114: 3865-3872Crossref PubMed Google Scholar). In the developing brain, β1-integrin is expressed in the ventricular zone (VZ) (4Campos L.S. Leone D.P. Relvas J.B. Brakebusch C. Fässler R. Suter U ffrench-Constant C. Development. 2004; 131: 3433-3444Crossref PubMed Scopus (267) Google Scholar) (Fig. 1C) by NSC that are exposed to a changing ECM and, possibly, to variable growth factor levels (13Campos L.S. BioEssays. 2005; 27: 698-707Crossref PubMed Scopus (94) Google Scholar, 29Campos L.S. J. Neurosci. Res. 2004; 78: 761-769Crossref PubMed Scopus (130) Google Scholar). Likewise, Notch1 plays a role in NSC and is thought to direct radial glial cell (RGC) differentiation (30Gaiano N. Nye J.S. Fishell G. Neuron. 2000; 26: 395-404Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 31Gaiano N. Fishell G. Annu. Rev. Neurosci. 2002; 25: 471-490Crossref PubMed Scopus (494) Google Scholar). Not surprisingly, Notch1 is expressed in the same VZ region (32Zhong W. Jiang M.M. Weinmaster G. Jan L.Y. Jan Y.N. Development. 1997; 124: 1887-1897Crossref PubMed Google Scholar) (Fig. 1B), and, interestingly, it is co-expressed with the β1-integrins (Fig. 1D). Co-expression of Notch1 and β1-integrins is also detectable in neurospheres (Fig. 1, E-G) (4Campos L.S. Leone D.P. Relvas J.B. Brakebusch C. Fässler R. Suter U ffrench-Constant C. Development. 2004; 131: 3433-3444Crossref PubMed Scopus (267) Google Scholar, 29Campos L.S. J. Neurosci. Res. 2004; 78: 761-769Crossref PubMed Scopus (130) Google Scholar, 33Capela A. Temple S. Neuron. 2002; 35: 865-875Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar) and in NSC/radial glial cell cultures, derived from ES cells (Fig. 1, H-J). The observation that both proteins are simultaneously expressed in neural progenitors and that their expression overlaps raises the hypothesis that they may cooperate or act in a coordinated fashion. To further test the hypothesis that β1-integrins and Notch pathways interact in neural progenitors/NSC, we used primary neurospheres and ES cell-derived NSC, the later providing an NSC-enriched population positive for RGC markers (Fig. 1, K and L) (21Bibel M. Richter J. Schrenk K. Tucker K.L. Staiger V. Nat. Neurosci. 2004; 7: 1003-1009Crossref PubMed Scopus (367) Google Scholar) (currently accepted to be NSC (34Alvarez-Buylla A. Garcia-Verdugo J.M. Tramontin A.D. Nat. Rev. Neurosci. 2001; 2: 287-293Crossref PubMed Scopus (845) Google Scholar)). Notch1 is highly expressed in the ES cell-derived NSC cultures and co-localizes with the β1-integrin (Fig. 1, H-J). The ES cell-derived NSC cultures are therefore suitable to study, in vitro, the cooperation between β1-integrin, growth factors, and Notch pathways, all of which are known to be crucial for NSC and RGC maintenance and development (4Campos L.S. Leone D.P. Relvas J.B. Brakebusch C. Fässler R. Suter U ffrench-Constant C. Development. 2004; 131: 3433-3444Crossref PubMed Scopus (267) Google Scholar, 31Gaiano N. Fishell G. Annu. Rev. Neurosci. 2002; 25: 471-490Crossref PubMed Scopus (494) Google Scholar, 35Forster E. Tielsch A. Saum B. Weiss K.H. Johanssen C. Graus-Porta D. Müller U. Frotscher M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13178-13183Crossref PubMed Scopus (222) Google Scholar).β1-Integrin and Growth Factor Receptors Are Required for Secondary Neurosphere Generation—To test the role of β1-integrin in NSC, we treated primary neurospheres with morpholino antisense oligonucleotides against β1-integrin to decrease the β1 subunit protein levels in EGF- or FGF-2-grown cells, prior to secondary neurosphere formation assays (see “Experimental Procedures”). The decrease in β1-integrin was confirmed by Western blot (Fig. 2E). Spheres treated with antisense or missense (control) morpholino oligonucleotides were dissociated and tested for their capacity to form new spheres (secondary neurosphere formation assay; see “Experimental Procedures”). These experiments show that a decrease in β1-integrin is associated with a moderate decrease in secondary neurosphere formation (Fig. 2, A-D). Interestingly, in EGF-grown (A and B) and FGF-2-grown (C and D) cells, the decrease in secondary neurosphere formation is more significant at low EGF levels (2 ng/ml; Fig. 2B) than at higher EGF levels (20 ng/ml; Fig. 2A), suggesting that in the presence of high EGF levels, the cells are less dependent on β1-integrin to maintain adequate levels of proliferation or survival. After morpholino treatment, the decrease in secondary neurosphere formation in FGF-2-grown cells (Fig. 2, C and D) is already apparent at high levels of FGF-2 (20 ng/ml; Fig. 2C) when compared with spheres grown with low levels of FGF-2 (2 ng/ml; Fig. 2D).FIGURE 2β1-integrins and EGFR are required for secondary neurosphere formation. A decrease in β1-integrin affects secondary neurosphere formation at low EGF and at high FGF-2 concentrations. Intact neurospheres were exposed to morpholino antisense to reduce β1-integrin expression and then dissociated and used in secondary neurosphere formation assays in the presence of 20 ng/ml EGF (A), 2 ng/ml EGF (B), 20 ng/ml FGF (C), and 2 ng/ml FGF (D). The experiments were carried out five times for each condition. A statistically significant decrease (p < 0.001) in the number of secondary neurospheres formed after antisense treatment was observed at 20 ng/ml EGF, 2 ng/ml EGF, and 20 ng/ml FGF-2. The strongest effect was seen for 20 ng/ml FGF-2 (C), followed in order by EGF 2 ng/ml (B) and EGF 20 ng/ml (A), suggesting that the morpholino-treated FGF-2-grown spheres are more dependent on β1-integrin than the EGF-grown ones. The decrease in β1-integrin was ascertained by Western blot (E). Samples grown in EGF or FGF-2 and either exposed to the morpholino (m) or to the missense (ms) were run in parallel after protein quantification. Note that the EGFR is markedly up-regulated at the nestin-rich edge of FGF-2-grown spheres, as detected by immunohistochemistry on sectioned neurospheres (F, nestin; G, EGFR) and confirmed with Western blot (H). The nuclei in F and G are labeled with DAPI (blue). To test the role of the EGFR activation in neural progenitor proliferation, intact mouse spheres (grown in a mix of EGF and FGF-2 for 8 days) were used for secondary neurosphere formation assays in the presence of FGF-2 and of an EGFR inhibitor (AG1478; 20 μm), as described under “Experimental Procedures” (J). Control cells were exposed to Me2SO alone. Exposure to the EGFR inhibitor induces a statistically significant reduction (p < 0.001, n = 3) in the number of secondary spheres formed, in the presence of FGF-2. Note that EGFR is highly expressed at the edge of neurospheres (G), in the region where nestin-positive progenitors are abundant (F). Exposure of neurospheres to growth factors affects NICD levels; the addition of EGF and FGF-2 to 24-h growth factor-starved neurospheres leads to an increase in detection of Notch intracellular domain (NICD) by Western blot (I). The experiment shown is representative of various replicates (n > 3). Note that as a result of EGF or FGF-2 addition (after growth factor starvation) MAPK is phosphorylated, whereas total levels of MAPK remain similar, indicating a strong and specific response to the growth factors. Equal amounts of proteins were loaded in each lane. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EGFR and β1-integrin interactions have been extensively demonstrated in three-dimensional breast culture systems (36Wang F. Weaver V.M. Petersen O.W. Larabell C.A. Dedhar S. Briand P. Lupu R. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14821-14826Crossref PubMed Scopus (547) Google Scholar), and it is conceivable that the high levels of EGFR found on the nestin-positive FGF-2-grown spheres (Fig. 2, F-H) may be responsible for the more acute response to a decrease in β1-integrin observed in FGF-2-grown cells. To test this hypothesis, spheres grown in both EGF and FGF-2 were used in a secondary neurosphere formation assay in the presence of FGF-2 and an EGFR inhibitor, AG1478 (20 μm). The exposure to AG1478 resulted in a sharp decrease in the number of secondary neurospheres formed, indicating that even for FGF-2-grown spheres, the EGFR is the crucial pathway involved in proliferation (Fig. 2J), as indeed suggested by the high levels of EGFR found on the FGF-2-grown cells (Fig. 2, G and H). Consequently, in FGF-2-grown spheres with decreased levels of β1-integrin (EGFR-strongly positive/β1-depleted), a lack of exposure to EGF will be severely felt (despite the high levels of EGFR expression) and cannot be compensated by (lacking) integrin activation. In the EGF-grown spheres (EGFR-positive/β1-depleted), even low levels of EGFR will be enough to respond to the EGF in the medium. These results suggest that β1-integrin may be important for EGFR activation in neurospheres and point toward a potential cooperation between the two pathways, as already described for epithelial cells and fibroblasts (37Kuwada S.K. Li X. Mol. Biol. Cell. 2000; 11: 2485-2496Crossref PubMed Scopus (112) Google Scholar, 38Moro L. Venturino M. Bozzo C. Silengo L. Altruda F. Beguinot L. Tarone G. Defilippi P. EMBO J. 1998; 17: 6622-6632Crossref PubMed Scopus (503) Google Scholar). Furthermore, it was recently shown that a decrease in β1-integrin causes a reduction in neurosphere size during secondary neurosphere formation assays, due to reduced progenitor proliferation and increased cell death (39Leone D.R.J. Campos L. Hemmi S. Brakebusch C. Fässler R. ffrench-Constant C. Suter U. J. Cell Sci. 2005; 118: 2589-2599Crossref PubMed Scopus (126) Google Scholar). The loss of β1-integrin in neurospheres also reduces the number of nestin-positive cells in a growth factor-dependent manner, and this phenotype can be rescued by exposing the cells to high growth factor levels (39Leone D.R.J. Campos L. Hemmi S. Brakebusch C. Fässler R. ffrench-Constant C. Suter U. J. Cell Sci. 2005; 118: 2589-2599Crossref PubMed Scopus (126) Google Scholar). This result is consistent with our morpholino experiments, and both may be explained by the signaling confluence of EGFR and β1-integrins toward the MAPK pathway. In fact, in the absence of β1-integrins, the signaling through the MAPK pathway may become more dependent on the presence of EGF in the medium (4Campos L.S. Leone D.P. Relvas J.B. Brakebusch C. Fässler R. Suter U ffrench-Constant C. Development. 2004; 131: 3433-3444Crossref PubMed Scopus (267) Google Scholar). Interestingly, we have also observed that the addition of EGF and FGF-2 to starved neurospheres leads to an increase of the detectable levels of NICD expression by Western blot (Fig. 2I). Other authors have observed that growth factors, such as ciliary neurotrophic factor, increase NICD expression levels (40Chojnacki A. Shimazaki T. Gregg C. Weinmaster G. Weiss S. J. Neurosci. 2003; 23: 1730-1741Crossref PubMed Google Scholar) or affect Notch activation (15Miyamoto Y. Maitra A. Ghosh B. Zechner U. Argani P. Iacobuzzo-Donahue C.A. Sriuranpong V. Iso T. Mezoely I.M. Wolfe M.S. Hruban R.H. Ball D.W. Schmid R.M. Leach S.D. Cancer Cell. 2003; 3: 565-576Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 41Hart A. Papadopoulou S. Edlund H. Dev. Dyn. 2003; 228: 185-193Crossref PubMed Scopus (165) Google Scholar). Taking into account that β1-integrins modulate the response to G" @default.
• W2020682158 created "2016-06-24" @default.
• W2020682158 creator A5058650953 @default.
• W2020682158 creator A5061430292 @default.
• W2020682158 creator A5073208795 @default.
• W2020682158 creator A5091468568 @default.
• W2020682158 date "2006-02-01" @default.
• W2020682158 modified "2023-09-26" @default.
• W2020682158 title "Notch, Epidermal Growth Factor Receptor, and β1-Integrin Pathways Are Coordinated in Neural Stem Cells" @default.
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