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Open Access Research

Hypothermia enhances induction of protective protein metallothionein under ischemia

Youn Hee Park1, Young Mi Lee1, Dong Sun Kim2, Jaechan Park3, Kyoungho Suk4, Jong Kun Kim5* and Hyung Soo Han1*

Author Affiliations

1 Department of Physiology, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Jung Gu, Daegu 700-422, Korea

2 Anatomy, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Daegu, Jung Gu, 700-422, Korea

3 Neurosurgery, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Daegu, Jung Gu, 700-422, Korea

4 Pharmacology, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Daegu, Jung Gu, 700-422, Korea

5 Emergency Medicine, Kyungpook National University School of Medicine, 101 Dongin 2 Ga, Jung Gu, Daegu 700-422, Korea

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Journal of Neuroinflammation 2013, 10:21  doi:10.1186/1742-2094-10-21

Published: 4 February 2013

Abstract

Background

Hypothermic protection against ischemic stroke has been reported by many studies. Hypothermia is supposed to mitigate the effects of deleterious genes and proteins and promote the activity of protective genes and proteins in the ischemic brain. Metallothionein (MT)-1/2 is thought to be a crucial factor for metal homeostasis, immune function, and apoptosis. This protein was found to exert protective effects in models of brain injury as well. In the present study, we investigated the effect of hypothermia on MT expression and the underlying mechanisms.

Methods

Cultured bEnd.3 brain endothelial cells were exposed to oxygen glucose deprivation and reperfusion (OGD+R). Reverse transcription PCR and western blot analyses were performed to measure the expression of MT, transcription factors, and methylation regulating factors. Transcription factor binding assays were also performed. Methylation profiles of the promoter area were obtained with pyrosequencing.

Results

Hypothermia protected bEnd.3 cells from OGD+R. When the cells were exposed to OGD+R, MT expression was induced. Hypothermia augmented MT levels. While OGD+R-induced MT expression was mainly associated with metal regulatory transcription factor 1 (MTF-1), MT expression promoted by hypothermia was primarily mediated by the signal transducer and activator of transcription 3 (STAT3). Significantly increased STAT3 phosphorylation at Ser727 was observed with hypothermia, and JSI-124, a STAT-3 inhibitor, suppressed MT expression. The DNA demethylating drug 5-aza-2-deoxycytidine (5-Aza) enhanced MT expression. Some of the CpG sites in the promoter MT=> it should be “the CpG sites in the MT promoter” showed different methylation profiles and some methylation regulating factors had different expressional profiles in the presence of OGD+R and hypothermia.

Conclusions

We demonstrated that hypothermia is a potent inducer of MT gene transcription in brain endothelial cells, and enhanced MT expression might contribute to protection against ischemia. MT gene expression is induced by hypothermia mainly through the STAT3 pathway. DNA methylation may contribute to MT gene regulation under ischemic or hypothermic conditions.

Keywords:
Hypothermia; Ischemia; STAT3; MRE; Methylation; Gene expression