title: Promotion of both proliferation and neuronal differentiation in pluripotent P19 cells with stable overexpression of the glutamine transporter slc38a1.
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.
  
  
  RESULTS
  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
  GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.
authors.affiliation: Laboratory of Molecular Pharmacology, Kanazawa University Graduate School, Kanazawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ogura
authors.forename: Takami
authors.initials: T
authors.lastname: Kakuda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takarada
authors.forename: Noritaka
authors.initials: N
authors.lastname: Nakamichi
authors.forename: Ryo
authors.initials: R
authors.lastname: Fukumori
authors.forename: Yeong-Hun
authors.initials: YH
authors.lastname: Kim
authors.forename: Eiichi
authors.initials: E
authors.lastname: Hinoi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D026924:Amino Acid Transport System A / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D054278:Embryonal Carcinoma Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D055495:Neurogenesis / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D026924:Amino Acid Transport System A; C432411:Slc38a1 protein, mouse