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Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine

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Abstract

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1–100 μM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

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Abbreviations

AP-1:

Activator protein-1

ATF2:

Activating transcription factor-2

ATF2:

Activating transcription factor-2

ATF3:

Activating transcription factor-3

ATRA:

All-trans retinoic acid

bHLH:

Basic helix-loop-helix

BrdU:

5-Bromo-2′-deoxyuridine

CaATF6:

Constitutive active activating transcription factor-6

CaHIF1α:

Constitutive active hypoxia inducible factor-1α

C/EBPα:

CCAAT enhancer binding protein-α

CNTF:

Ciliary neurotrophic factor

CNTFRα:

Ciliary neurotrophic factor receptor alpha

CREB:

Cyclic AMP responsive element binding protein

DMEM/F-12:

Dulbecco’s modified Eagle medium: Nutrient Mixture F-12

ER:

Endoplasmic reticulum

EV:

Empty vector

FBS:

Fetal bovine serum

FGF:

Fibroblast growth factor

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GFAP:

Glial fibrillary acidic protein

Gln:

Glutamine

HIF1α:

Hypoxia inducible factor-1α

MAP2:

Microtubules-associated protein-2

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NCAM:

Neural cell adhesion molecule

NFAT2:

Nuclear factor of activated T cell-1

NF-κB:

Nuclear factor-κB

Nrf2:

Nuclear factor (erythroid-derived 2)-like 2

PA:

Paraformaldehyde

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

RARα:

Retinoic acid receptor alpha

RT-PCR:

Reverse transcription polymerase chain reaction

SLC38A1:

Solute carrier 38a1

STAT1:

Signal transducer and activator of transcription-1

STAT3:

Signal transducer and activator of transcription-3

WT:

Wild-type

XBP1:

X-box binding protein-1

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Acknowledgments

This work was in part supported by Grants-in-Aids for Scientific Research to TT (No. 22500330) and YY (Nos. 18053009 and 24650196) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of interest

None.

Author information

Author notes

  1. Masato Ogura

    Present address: Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan

  2. Noritaka Nakamichi

    Present address: Laboratory of Molecular Pharmacotherapeutics, Kanazawa University Graduate School, Kanazawa, 920-1192, Japan

Authors and Affiliations

  1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan

    Takeshi Takarada, Masato Ogura, Noritaka Nakamichi, Ryota Nakazato, Hiroshi Kokubo, Shinsuke Ikeno, Saki Nakamura, Takaya Kutsukake, Eiichi Hinoi & Yukio Yoneda

  2. Research Center of Composite Material, Fukuoka University, Fukuoka, 814-0180, Japan

    Takami Kakuda

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  1. Takeshi Takarada
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Correspondence to Yukio Yoneda.

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Special Issue: 40th Year of Neurochemical Research.

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Takarada, T., Ogura, M., Nakamichi, N. et al. Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine. Neurochem Res 41, 5–15 (2016). https://doi.org/10.1007/s11064-015-1591-4

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  • DOI: https://doi.org/10.1007/s11064-015-1591-4

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