Abstract
ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.
[1] Lindvall, O., Kokaia, Z. and Martinez-Serrano, A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat. Med. 10Suppl (2004) S42–50. http://dx.doi.org/10.1038/nm106410.1038/nm1064Search in Google Scholar PubMed
[2] Clelland, C.D., Barker, R.A. and Watts, C. Cell therapy in Huntington disease. Neurosurg. Focus 24 (2008) E9. http://dx.doi.org/10.3171/FOC/2008/24/3-4/E810.3171/FOC/2008/24/3-4/E8Search in Google Scholar PubMed
[3] Locatelli, F., Bersamo, A., Ballabio, E., Lanfranconi, S., Papdimitriou, D., Strazzer, S., Bresolin, N., Comi, G.P. and Corti, S. Stem cell therapy in stroke. Cell. Mol. Life Sci. 66 (2009) 757–772. http://dx.doi.org/10.1007/s00018-008-8346-110.1007/s00018-008-8346-1Search in Google Scholar PubMed
[4] Donato, R., Miljan, E.A., Hines, S.J., Aouabdi, S., Pollock, K., Patel, S., Edwards, F.A. and Sinden, J.D. Differential development of neuronal physiological responsiveness in two human neural stem cell lines. BMC Neurosci. 8 (2007) 36. http://dx.doi.org/10.1186/1471-2202-8-3610.1186/1471-2202-8-36Search in Google Scholar PubMed PubMed Central
[5] Hoffrogge, R., Mikkat, S., Scharf, C., Beyer, S., Christoph, H., Pahnke, J., Mix, E., Berth, M., Uhrmacher, A., Zubrzycki, I.Z., Miljan, E., Völker, U. and Rolfs, A. 2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM). Proteomics 6 (2006) 1833–1847. http://dx.doi.org/10.1002/pmic.20050055610.1002/pmic.200500556Search in Google Scholar PubMed
[6] Morgan, P.J., Ortinau, S., Frahm, J., Kruger, N., Rolfs, A. and Frech, M.J. Protection of neurons derived from human neural progenitor cells by veratridine. Neuroreport 20 (2009) 1225–1229. http://dx.doi.org/10.1097/WNR.0b013e32832fbf4910.1097/WNR.0b013e32832fbf49Search in Google Scholar PubMed
[7] Logan, C.Y. and Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20 (2004) 781–810. http://dx.doi.org/10.1146/annurev.cellbio.20.010403.11312610.1146/annurev.cellbio.20.010403.113126Search in Google Scholar PubMed
[8] Komiya, Y. and Habas, R. Wnt signal transduction pathways. Organogenesis 4 (2008) 68–75. http://dx.doi.org/10.4161/org.4.2.585110.4161/org.4.2.5851Search in Google Scholar PubMed PubMed Central
[9] Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N. and Gotoh, Y. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131 (2004) 2791–2801. http://dx.doi.org/10.1242/dev.0116510.1242/dev.01165Search in Google Scholar PubMed
[10] Mutoyama, Y., Kondoh, H. and Takada, S. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochem. Biophys. Res. Commun. 313 (2004) 915–921. http://dx.doi.org/10.1016/j.bbrc.2003.12.02310.1016/j.bbrc.2003.12.023Search in Google Scholar PubMed
[11] Castelo-Branco, G., Wagner, J., Rodriguez, F.J., Kele, J., Sousa, K., Rawal, N., Pasolli, H.A., Fuchs, E., Kitajewski, J. and Arenas, E. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad. Sci. USA 100 (2003) 12747–12752. http://dx.doi.org/10.1073/pnas.153490010010.1073/pnas.1534900100Search in Google Scholar
[12] Kholodenko B.N. Cell-signaling dynamics in time and space. Nat. Rev. Mol. Cell. Biol. 7 (2006) 165–176. http://dx.doi.org/10.1038/nrm183810.1038/nrm1838Search in Google Scholar
[13] Nakamura, T., Sano, M., Songyang, Z. and Schneider, M.D. A Wnt- and beta-catenin-dependent pathway for mammalian cardiac myogenesis. Proc. Natl. Acad. Sci. USA 100 (2003) 5834–5839. http://dx.doi.org/10.1073/pnas.093562610010.1073/pnas.0935626100Search in Google Scholar
[14] Hübner, R., Schmöle, A.C., Liedmann, A., Frech, M.J., Rolfs, A. and Luo, J. Differentiation of human neural progenitor cells regulated by Wnt-3a. Biochem. Biophys. Res. Commun. 400 (2010) 358–362. http://dx.doi.org/10.1016/j.bbrc.2010.08.06610.1016/j.bbrc.2010.08.066Search in Google Scholar
[15] Schmöle, A.C., Brenführer, A., Karapetyan, G., Jaster, R., Pews_Davtyan, A., Hübner, R., Ortinau, S., Beller, M., Rolfs, A. and Frech, M.J. Novel indolylmaleimide acts as GSK-3β inhibitor in human neural progenitor cells. Bioorg. Med. Chem. 18 (2010) 6785–6795. http://dx.doi.org/10.1016/j.bmc.2010.07.04510.1016/j.bmc.2010.07.045Search in Google Scholar
[16] Klipp, E. and Liebermeister, W. Mathematical modeling of intracellular signalling pathways. BMC Neuroscience 7 (2006) S10. http://dx.doi.org/10.1186/1471-2202-7-S1-S1010.1186/1471-2202-7-S1-S10Search in Google Scholar
[17] Mazemondet, O., John, M., Maus, C., Uhrmacher A. and Rolfs, A. Integrating diverse reaction types into stochastic models — a signaling pathway case study in the imperative pi-Calculus. In: Proceedings of Winter Simulation Conference, 2009, 931–943. 10.1109/WSC.2009.5429723Search in Google Scholar
[18] Pfaffl, M.W. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29 (2001) e45. http://dx.doi.org/10.1093/nar/29.9.e4510.1093/nar/29.9.e45Search in Google Scholar
[19] Ohl, F., Jung, M., Radonic, A., Sachs, M., Loening, S.A. and Jung, K. Identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer. J. Urol. 175 (2006) 1915–1920. http://dx.doi.org/10.1016/S0022-5347(05)00919-510.1016/S0022-5347(05)00919-5Search in Google Scholar
[20] Schiling, M., Maiwald, T., Bohl, S., Kollmann, M., Kreuts, C., Timmer, J. and Klinmüller, U. Computational processing and error reduction strategies for standardized quantitative data in biological networks. FEBS J. 272 (2005) 6400–6411. http://dx.doi.org/10.1111/j.1742-4658.2005.05037.x10.1111/j.1742-4658.2005.05037.xSearch in Google Scholar PubMed
[21] Angers, S. and Moon, R.T. Proximal events in Wnt signal transduction. Nat. Rev. Mol. Cell Biol. 10 (2009) 468–477. 10.1038/nrm2717Search in Google Scholar
[22] Stambolic, V., Ruel, L. and Woodgett, J.R. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol. 6 (1996) 1664–1668. http://dx.doi.org/10.1016/S0960-9822(02)70790-210.1016/S0960-9822(02)70790-2Search in Google Scholar
[23] Jho, E.H., Zhang, T., Domon, C., Joo, C.K., Freund, J.N. and Costantini, F. Wnt/beta-catenin/Tcf signalling induces the transcription of Axin2, a negative regulator of the signalling pathway. Mol. Cell Biol. 22 (2002) 1172–1183. http://dx.doi.org/10.1128/MCB.22.4.1172-1183.200210.1128/MCB.22.4.1172-1183.2002Search in Google Scholar
[24] Blagosklonny, M.V. and Pardee, A.B. The restriction point of the cell cycle. Cell Cycle 1 (2002) 103–110. Search in Google Scholar
[25] Li, Y., Wenyan L., Xi, H. and Guojun, B. Modulation of LRP6-mediated Wnt signaling by molecular chaperone Mesd. FEBS Lett. 580 (2006) 5423–5428. http://dx.doi.org/10.1016/j.febslet.2006.09.01110.1016/j.febslet.2006.09.011Search in Google Scholar
[26] Tamai, K., Zeng, X., Liu, C., Zhang, X., Harada, Y., Chang, Z. and He, X. A mechanism for Wnt coreceptor activation. Mol. Cell 13 (2004) 149–156. http://dx.doi.org/10.1016/S1097-2765(03)00484-210.1016/S1097-2765(03)00484-2Search in Google Scholar
[27] Khan, Z., Vijayakumar, S., De La Torre, T.V., Rotolo, S. and Bafico, A. Analysis of endogenous LRP6 function reveals a novel feedback mechanism by which Wnt negatively regulates its receptor. Mol. Cell Biol. 27 (2007) 7291–7301. http://dx.doi.org/10.1128/MCB.00773-0710.1128/MCB.00773-07Search in Google Scholar
[28] Winer, J., Jung, C.K., Shackel, I. and Williams, P.M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270 (1999) 41–49. http://dx.doi.org/10.1006/abio.1999.408510.1006/abio.1999.4085Search in Google Scholar
[29] Willems, E., Mateizel, I., Kemp, C., Gauffman, G., Sermon, K. and Leyns, L. Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells. Int. J. Dev. Biol. 50 (2006) 627–635. http://dx.doi.org/10.1387/ijdb.052130ew10.1387/ijdb.052130ewSearch in Google Scholar
[30] Semenov, M.V., Tamai, K., Brott, B.K., Kuehl, M., Sokol, S. and He, X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr. Biol. 11 (2001) 951–961. http://dx.doi.org/10.1016/S0960-9822(01)00290-110.1016/S0960-9822(01)00290-1Search in Google Scholar
[31] Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., Walter, C., Glinka, A. and Niehrs, C. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417(6889) (2004) 664–667. http://dx.doi.org/10.1038/nature75610.1038/nature756Search in Google Scholar PubMed
[32] Semenov, M.V., Zhang, X. and He, X. DKK1 antagonizes Wnt signaling without promotion of LRP6 internalization and degradation. J. Biol. Chem. 283 (2008) 21427–21432. http://dx.doi.org/10.1074/jbc.M80001420010.1074/jbc.M800014200Search in Google Scholar PubMed PubMed Central
[33] Yoshikawa, H., Matsubara, K., Zhou, X., Okumara, S., Kubo, T., Murase, Y., Shikauchi, Y., Esteller, M., Herman, J.G., Wei, X. and Harris, C.C. WNT10B functional dualism: beta-catenin/Tcf-dependent growth promotion or independent suppression with deregulated expression in cancer. Mol. Biol. Cell 18 (2007) 4292–4303. http://dx.doi.org/10.1091/mbc.E06-10-088910.1091/mbc.e06-10-0889Search in Google Scholar PubMed PubMed Central
[34] Kirikoshi, H. and Katoh, M. Expression and regulation of WNT10B in human cancer: up-regulation of WNT10B in MCF-7 cells by beta-estradiol and down-regulation of WNT10B in NT2 cells by retinoic acid. Int. J. Mol. Med. 10 (2002) 507–511. Search in Google Scholar
[35] Ishikawa, T., Tamai, Y., Zorn, A.M., Yoshida, H., Seldin, M.F., Nishikawa, S. and Taketo, M.M. Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placenta angiogenesis. Development 128 (2001) 25–33. Search in Google Scholar
[36] Snow, G.E., Kasper, A.C., Busch, A.M., Schwarz, E., Ewings, K.E., Bee, T., Spinella, M.J., Dmitrovsky, E. and Freemantle, S.J. Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting. BMC Cancer 9 (2009) 83. http://dx.doi.org/10.1186/1471-2407-9-38310.1186/1471-2407-9-383Search in Google Scholar PubMed PubMed Central
[37] van Amerongen, R., Mikels, A. and Nusse, R. Alternative Wnt signaling is initiated by distinct receptors. Sci. Signal. 1 (2008) re9. http://dx.doi.org/10.1126/scisignal.135re910.1126/scisignal.135re9Search in Google Scholar PubMed
[38] Caricasole, A., Ferraro, T., Iacovelli, L., Barletta, E., Caruso, A., Melchiorri, D., Terstappen G.C. and Nicoletti, F. Functional characterization of WNT7A signaling in PC12 cells: interaction with a FZD5-LRP6 receptor complex and modulation by Dickkopf proteins. J. Biol. Chem. 278 (2003) 37024–37031. http://dx.doi.org/10.1074/jbc.M30019120010.1074/jbc.M300191200Search in Google Scholar PubMed
[39] Carmon, K.S. and Loose, D.S. Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells. Mol. Cancer Res. 6 (2008) 1017–1028. http://dx.doi.org/10.1158/1541-7786.MCR-08-003910.1158/1541-7786.MCR-08-0039Search in Google Scholar PubMed
[40] Le Grand, F., Jones, A.E., Seale, V., Scime, A. and Rudnicki, M.A. Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem Cell 4 (2009) 535–547. http://dx.doi.org/10.1016/j.stem.2009.03.01310.1016/j.stem.2009.03.013Search in Google Scholar PubMed PubMed Central
[41] Yang, Y., Topol, L., Lee, H. and Wu, J. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130 (2003) 1003–1015. http://dx.doi.org/10.1242/dev.0032410.1242/dev.00324Search in Google Scholar PubMed
[42] Castelo-Brance, G., Sousa, K.M., Bryja, V., Pinto, L., Wagner, J. and Arenas, E. Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion. Mol. Cell Neurosci. 31 (2006) 251–262. http://dx.doi.org/10.1016/j.mcn.2005.09.01410.1016/j.mcn.2005.09.014Search in Google Scholar PubMed
[43] Beagle, B., Mi, K. and Johnson, G.V.W. Phosphorylation of PPP(S/Y)P motif of the free LRP6 intracellular domains is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity. J. Cell Biochem. 108 (2009) 886–895. http://dx.doi.org/10.1002/jcb.2231810.1002/jcb.22318Search in Google Scholar PubMed PubMed Central
[44] Wu, G., Huang, H., Abreu, J.G. and He, X. Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6. PloS One 4 (2009) e4926. http://dx.doi.org/10.1371/journal.pone.000492610.1371/journal.pone.0004926Search in Google Scholar PubMed PubMed Central
[45] Bryja, V., Schulte, G. and Arenas, E. Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin. Cell Signal. 19 (2007b) 610–616. http://dx.doi.org/10.1016/j.cellsig.2006.08.01110.1016/j.cellsig.2006.08.011Search in Google Scholar PubMed
[46] Yokoyama, N., Yin, D. and Malbon, C.C. Abundance, complexation, and trafficking of Wnt/beta-catenin signaling elements in response to Wnt3. J. Mol. Signal. 2 (2007) 11. http://dx.doi.org/10.1186/1750-2187-2-1110.1186/1750-2187-2-11Search in Google Scholar PubMed PubMed Central
[47] Müller, H.A., Samanta, R. and Wieschaus, E. Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes. Development 126 (1999) 577–586. Search in Google Scholar
[48] Cadigan, K.M. and Nusse, R. Wnt signaling: a common theme in animal development. Genes Dev. 11 (1997) 3286–3305. http://dx.doi.org/10.1101/gad.11.24.328610.1101/gad.11.24.3286Search in Google Scholar PubMed
[49] Sato, A., Kojima, T., Ui-Tei, K., Miyata, Y. and Saigo, K. Frizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants. Development 126 (1999) 4421–4430. Search in Google Scholar
[50] Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H., Schlag, P.M., Birchmeier, W. and Behrens, J. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell Biol. 22 (2004) 1184–1193. http://dx.doi.org/10.1128/MCB.22.4.1184-1193.200210.1128/MCB.22.4.1184-1193.2002Search in Google Scholar PubMed PubMed Central
© 2011 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.