Abstract
The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.
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This work was supported by the polish Ministry of Science and Higher Education grant no. N N401 063636.
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Editor: T. Okamoto
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Supplementary figure 1
Dose-dependent Dox-induced expression of trkC gene in clone 4.1. Cells were treated for 24 h with different Dox concentrations. Relative quantification of Dox-induced trkC mRNAs were determined by real-time PCR with SYBR Green and normalized to the hprt mRNAs. Data are representative from 4 independent experiments. (GIF 41 kb)
Supplementary figure 2
Expression of exogenous TrkC protein in clone 4.1. Cells were grown in the absence (A, B) or presence of 1 μg/mL Dox (C). Cells were immunostained for TrkC (red) with goat anti-TrkC primary antibody (B, C) plus anti-goat IgG secondary antibody conjugated to Cy3 (A-C). Cells were stained for nuclei (green) with YO-PRO-1 iodide and analyzed by immunofluorescence microscopy. Data are representative from 2 independent experiments. (GIF 82 kb)
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Krawczyk, P., Twarog, E., Kurowska, E. et al. Establishment of a cellular model to study TrkC-dependent neuritogenesis. In Vitro Cell.Dev.Biol.-Animal 51, 241–248 (2015). https://doi.org/10.1007/s11626-014-9829-z
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DOI: https://doi.org/10.1007/s11626-014-9829-z