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
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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)