Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC-5 Cells

Jong Eun Woo; Min Young Kwon; Su Wol Chung; Je Moon Woo

doi:10.3341/jkos.2014.55.8.1195

Journal List > J Korean Ophthalmol Soc > v.55(8) > 1009753

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Woo, Kwon, Chung, and Woo: Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC-5 Cells

Original Article

J Korean Ophthalmol Soc 2014;55(8):1195-1201.

Published online: 31 August 2014

DOI: https://doi.org/10.3341/jkos.2014.55.8.1195

Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC-5 Cells

Jong Eun Woo, MD¹, Min Young Kwon², Su Wol Chung, PhD², Je Moon Woo, MD, PhD¹

¹Department of Ophthalmology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea

²School of Biological Sciences, College of Natural Science, University of Ulsan, Ulsan, Korea

Address reprint requests to Je Moon Woo, MD, PhD, Department of Ophthalmology, Ulsan University Hospital, #877 Bangeojinsunhwando-ro, Dong-gu, Ulsan 682-714, Korea, Tel: 82-52-250-7177, Fax: 82-52-250-7174, E-mail: limbus68@naver.com

Received 28 September 2013 Revised 12 February 2014 Accepted 24 June 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

In order to determine whether the Tonicity responsive enhancer binding protein (TonEBP) is expressed by hypertonic and hyperosmolar stress, TonEBP expression was investigated in the retinal ganglion cell (RGC) line, RGC-5 cells.

Methods

After RGC-5 cells were cultured by Staurosporine, TonEBP expression was measured with Western immunoblotting analysis and real-time reverse transcription-polymerase chain reaction in 50 mM NaCl, 100 mM mannitol, 50 mM glucose, or 100 mM glucose at 3, 6, 12, and 24 hours after exposure to each environment.

Results

In this study, the protein expression of TonEBP was determined to be statistically significantly checked in 50 mM NaCl after 3, and 6 hours, in 100 mM mannitol after 6 hours, and in 100 mM glucose after 3, and 6 hours. TonEBP messenger Ribonucleic acid (mRNA) expression was determined to be statistically significantly checked in 50 mM NaCl after 3 hours, in 100 mM mannitol after 3, and 24 hours, and in 50 mM glucose after 3, and 24 hours.

Conclusions

These results suggested that TonEBP was expressed by hypertonic and hyperosmolar stress at the protein and mRNA levels. Further studies are nedded to determine the role of TonEBP and the mechanism of expression and regulation of TonEBP.

Keywords: Hyperosmolar stress, Hypertonic stress, Retinal ganglion cell death, TonEBP expression

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Figure 1.

Hypertonic inducers increase Tonicity responsive enhancer binding protein (TonEBP) protein expression in RGC-5 cells (A, C: 50 mM NaCl; B, D: 100 mM mannitol; -STD: before preconditioning; V: just after the preconditioning; 3: 3 hours after preconditioning; 6: 6 hours after preconditioning; 12: 12 hours after preconditioning; 24: 24 hours after preconditioning; ^*p< 0.05 by Mann-Whitney U -test). RGC = retinal ganglion cell; STD = staurosporine; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

High glucose increases Tonicity responsive enhancer binding protein (TonEBP) protein expression in RGC-5 cells (50 mM glucose, 100 mM glucose; A: Western blotting; B: Histogram; -STD: before preconditioning; V: just after the preconditioning; 3: 3 hours after preconditioning; 6: 6 hours after preconditioning; 12: 12 hours after preconditioning; 24: 24 hours after preconditioning; ^*p < 0.05 by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

Hypertonic inducers increase Tonicity responsive enhancer binding protein (TonEBP) messenger Ribonucleic acid (mRNA) expression in RGC-5 cells (A: 50 mM NaCl; B: 100 mM mannitol; -STD: before preconditioning; +STD and -NaCl/-mannitol: just after the preconditioning; 3: 3 hours after preconditioning; 6: 6 hours after preconditioning; 12: 12 hours after preconditioning; 24: 24 hours after preconditioning; ^*p < 0.05 by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine.

Figure 4.

High glucose increases Tonicity responsive enhancer binding protein (TonEBP) messenger Ribonucleic acid (mRNA) expression in RGC-5 cells (-STD: before preconditioning; +STD and -glucose: just after the preconditioning; 3: 3 hours after preconditioning; 6: 6 hours after preconditioning; 12: 12 hours after preconditioning; 24: 24 hours after preconditioning; ^*p < 0.05 by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine.

Table 1.

Primer sequences for real-time PCR

Gene name	Sequence
NFAT5	F 5’-TTC ATC TCA TTG CTC AGC G-3’
(TonEBP)	R 5’-GGG AGA AGA TCA TAG ACA GAT TC-3’
GAPDH	F 5’-GGC ACA GTC AAG GCT GAG AAT G-3’
GAPDH	R 5’-ATG GTG GTG AAG ACG CCA GTA-3’

F = forward; R = reverse; PCR = polymerase chain reaction; NFAT5 = nuclear factor of activated T-cells 5; TonEBP = tonicity responsive enhancer binding protein; GAPDH = glyceraldehyde- 3-phosphate dehydrogenase.

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