Korean J Physiol Pharmacol.  2015 Mar;19(2):105-109. 10.4196/kjpp.2015.19.2.105.

NgR1 Expressed in P19 Embryonal Carcinoma Cells Differentiated by Retinoic Acid Can Activate STAT3

• Lee SI
• Yun J
• Baek JY
• Jeong YJ
• Kim JA
• Kang JS
• Park SH
• Kim SK
• Park SK

• KMID: 2285561
• DOI: http://doi.org/10.4196/kjpp.2015.19.2.105

Abstract

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

Keyword

Neuronal differentiation; NMDA receptor; Nogo receptor; P19 cell; STAT3

MeSH Terms

Axons
Central Nervous System
Embryonal Carcinoma Stem Cells*
Mass Screening
N-Methylaspartate
Neurites
Neurons
Phosphorylation
Plastics
Receptors, N-Methyl-D-Aspartate
Regeneration
RNA, Messenger
Tretinoin*
N-Methylaspartate
Plastics
RNA, Messenger
Receptors, N-Methyl-D-Aspartate
Tretinoin

Figure

• 

  Fig. 1 Differentiation of P19 embryonal carcinoma cells by all trans-retinoic acid. (A) Morphology of the parental P19 cells before differentiation. (B) Morphology of the cells differentiated by 0.5 µM all trans-retinoic acid, as described in "METHODS".

• 

  Fig. 2 Expression of NgR1 mRNA and protein by the differentiated P19 cells. (A) and (B) RT-PCR and Western immunoblot analyses for NgR1 expression, respectively. BD, before differentiation; EB, embryonic bodies; D3 and D5, the 3rd and 5th day of differentiation.

• 

  Fig. 3 Expression of mRNA of NMDA receptor subtypes in neuronally differentiated P19 cells and SH-SY5Y cells. (A) Differentiation of P19 cells were induced by all trans-retinoic acid and the expression of mRNA of mouse NR1, NR2A, NR2B, NR2C, and NR2D were detected by RT-PCR analysis. BD, before differentiation; EB, embryonic bodies; D1~D5, the 1st~5th day of differentiation. (B) Differentiation of SH-SY5Y cells were induced by all trans-retinoic acid alone or sequential combination of all trans-retinoic acid and BDNF. The expression of mRNA of human NR1, NR2A, NR2B, NR2C, and NR2D were detected by RT-PCR analysis. BD, before differentiation; RA, 10 µM all trans-retinoic acid; BDNF, 25 ng/ml brain derived neurotrophic factor.

• 

  Fig. 4 Increased phosphorylation of STAT3 by P4 peptide in neuronally differentiated P19 cells. P19 cells differentiated for 5 days were incubated in the presence and absence of Nogo-P4 peptide and/or NEP1-40 for 30 min and the cell lysates were applied to western immunoblotting for STAT3, phospho-STAT3 (p-STAT3), mTOR and phospho-mTOR (p-mTOR). (A) Representative images of western immunoblotting are shown. (B) Densitometry results of p-STAT3 are expressed as mean±SD (n=3, *p<0.05). Values shown in the graph represent the ratio of density of p-STAT3 and β-actin. Lane 1: control; lane 2: 4 µM Nogo-P4; lane 3: 4 µM Nogo-P4+2 µM NEP1-40; lane 4: 2 µM NEP1-40.

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