RRC ID 61957
Author Ogura M, Kakuda T, Takarada T, Nakamichi N, Fukumori R, Kim YH, Hinoi E, Yoneda Y.
Title Promotion of both proliferation and neuronal differentiation in pluripotent P19 cells with stable overexpression of the glutamine transporter slc38a1.
Journal PLoS One
Abstract BACKGROUND:We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.
METHODOLOGY/PRINCIPAL FINDINGS:The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.
CONCLUSIONS/SIGNIFICANCE:GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.
Volume 7(10)
Pages e48270
Published 2012-1-1
DOI 10.1371/journal.pone.0048270
PII PONE-D-12-14189
PMID 23110224
PMC PMC3480496
MeSH Amino Acid Transport System A / genetics Amino Acid Transport System A / metabolism* Animals Blotting, Northern Cell Differentiation / genetics Cell Differentiation / physiology* Cell Line, Tumor Cell Proliferation Chromatography, High Pressure Liquid Embryonal Carcinoma Stem Cells / cytology* Embryonal Carcinoma Stem Cells / metabolism* Immunohistochemistry Mice Neurogenesis / genetics Neurogenesis / physiology* Reverse Transcriptase Polymerase Chain Reaction
IF 2.74
Human and Animal Cells P19CL6