Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation

Cellular & Molecular Biology Letters, Dec 2011

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.

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Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation

Volume QUANTITATIVE AND KINETIC PROFILE OF Wnt/β-CATENIN SIGNALING COMPONENTS DURING HUMAN NEURAL PROGENITOR CELL DIFFERENTIATION ORIANNE MAZEMONDET 2 3 RAYK HUBNER 2 JANA FRAHM 2 DIRK KOCZAN 1 2 BENJAMIN M. BADER 0 2 DIETER G. WEISS 0 2 ADELINDE M. UHRMACHER 2 3 MORITZ J. FRECH 2 ARNDT ROLFS 2 JIANKAI LUO jiankai.luo@uni- 2 Albrecht-Kossel-Institute for Neuroregeneration 2 Centre for Mental Health 2 0 Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock , 18059 Rostock , Germany 1 Proteome Center Rostock, University of Rostock , Schillingallee 69, 18055 Rostock , Germany 2 Disease, University of Rostock , Gehlsheimer Strasse 20, 18147 Rostock , Germany 3 Modelling and Simulation Group, Institute of Computer Science, University of Rostock , Albert-Einstein-Str. 21, 18059 Rostock , Germany 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, § Both authors contributed equally to this work and should be considered co-first authors - Abbreviations used: APC – adenomatous polyposis coli; bFGF – basic fibroblast growth factor; Cdk – cyclin-dependent kinase; CK1 – casein kinase 1; DAPI – 4',6-diamidino-2phenylindole; Dkk1 – Dickkopf 1; DMEM – Dulbecco’s modified Eagle’s medium; Dvl – dishevelled; EGF – epidermal growth factor; Fz – Frizzled; GAPDH – glyceraldehyde 3phosphate dehydrogenase; GFP – green fluorescent protein; GSK3beta – glycogen synthase kinase 3; HBSS – Hank’s buffered salt solution; hNPC – human neural progenitor cell; LRP6 – low-density lipoprotein receptor-related protein 6; MAP2 – microtubuleassociated protein 2; NPC – neural progenitor cell; Ror2 – receptor tyrosine kinase-like orphan receptor 2; Ryk – receptor-like tyrosine kinase; TCF – T-cell factor Human neural progenitor cells (hNPCs) are considered as a therapeutic tool for treatment of neurological diseases such as Parkinson’s and Huntington’s diseases, spinal cord injury and stroke [ 1-3 ]. hNPCs can be first differentiated, in vitro, into the desired type of neurons, which are sequentially used for transplantation treatment [ 1 ]. ReNcell VM [ 4 ], a hNPC line derived from the ventral mesencephalon of a 10-week old fetus, has been extensively characterized and is known to differentiate, in vitro, into astrocytes, oligodendrocytes and neurons, e.g., dopaminergic neurons [ 4-6 ]. Wnt proteins are secreted morphogens involved in cell proliferation and differentiation, cellular polarity and apoptosis, cell migration, axon guidance, and synaptogenesis, and play an important role during embryogenesis and tissue formation [ 7 ]. Upon binding to Frizzled (Fz) receptors on the cell surface, Wnt proteins can trigger three different signaling pathways: the Wnt/β-catenin pathway, the Wnt/calcium pathway, and the planar cell polarity (PCP) pathway. In the Wnt/β-catenin pathway, a series of events occurs when Wnt proteins bind to the Fzs and their co-receptors LRP5/6, resulting in a stabilization of β-catenin in the cytosol by activation of Dishevelled (Dvl) family proteins. The Dvl proteins inhibit the degradation complex, which consists of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), resulting in accumulation of β-catenin in the cytosol. Subsequently, β-catenin translocates into the nucleus, where it interacts with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors to drive specific expression of target genes [ 8 ]. Remarkably, Wnt signals regulate both proliferation and differentiation of NPCs in a stage-specific and cellular contextdependent manner [ 9, 10 ]. For example, Wnt1, Wnt3a and Wnt5a cooperate and regulate the proliferation and differentiation of dopaminergic progenitor cells during neurogenesis in the ventral midbrain [11]. However, little is known about the kinetic regulation of the Wnt signals during these processes although the specificity of signaling pathways rely on their temporal and spatial dynamics, especially of the pathway’s downstream signaling proteins [ 12 ]. Our previous studies showed that the Wnt/β-catenin pathway and Wnt3a, a typical activator of the Wnt/β-catenin pathway [ 13 ], regulate neurogenesis of ReNcell VM cells in vitro [ 14, 15 ]. Thus, in the present study, we investigate the kinetic and dynamic Wnt/β-catenin pathway during physiological differentiation of ReNcell VM cells, in order to elucidate (...truncated)


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Orianne Mazemondet, Rayk Hubner, Jana Frahm, Dirk Koczan, Benjamin M. Bader, Dieter G. Weiss, Adelinde M. Uhrmacher, Moritz J. Frech, Arndt Rolfs, Jiankai Luo. Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation, Cellular & Molecular Biology Letters, 2011, pp. 515, Volume 16, Issue 4, DOI: 10.2478/s11658-011-0021-0