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. 2012 Mar 7;17(2):309–322. doi: 10.2478/s11658-012-0012-9

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation

Shian-Jang Yan 1, Yi-Fen Lee 1, Huei-Ju Ting 1, Ning-Chun Liu 1, Su Liu 1, Shin-Jen Lin 1, Shauh-Der Yeh 2, Gonghui Li 1, Chawnshang Chang 1,3,✉
PMCID: PMC3402907  NIHMSID: NIHMS388235  PMID: 22396141

Abstract

The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.

Key words: TR4, GADD45A, Ionizing radiation, Mouse embryonic fibroblast, Genotoxic stress, TR4 response element

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Abbreviations used

ChIP

chromatin immunoprecipitation

DR

direct repeat

GADD45A

growth arrest and DNA-damage-inducible, alpha

GADDLuc

GADD45A gene controlled luciferase reporter

IgG

immunoglobulin G

IR

ionizing radiation

MEF

mouse embryonic fibroblast

MTT

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

SC

cells expressing scramble control shRNA

shRNA

small hairpin RNA

shTR4

cell expressing shRNA targeting TR4

TR4

testicular nuclear receptor 4

TR4KO

TR4 knockout

TR4RE

TR4 response element

UV

ultraviolet light irradiation

WT

wild type

Footnotes

These authors contributed equally to this paper.

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