Investigating transcriptional regulation of Prdx6 in mouse liver cells

doi:10.1016/j.freeradbiomed.2007.01.023

Free Radical Biology and Medicine

Volume 42, Issue 8, 15 April 2007, Pages 1270-1277

https://doi.org/10.1016/j.freeradbiomed.2007.01.023 Get rights and content

Abstract

Prdx6, a unique member of the peroxiredoxin family of antioxidants, is highly expressed in liver and protects cells from oxidative damage by reducing H₂O₂ and various lipid peroxides. We investigated the transcriptional regulation of Prdx6 in the H2.35 mouse hepatocyte cell line and sought to determine the mechanism of basal and induced expression. We found that Prdx6 expression is down-regulated upon serum deprivation and subsequently induced in a time-dependent manner in response to KGF, TNF-α, dexamethasone, and H₂O₂. Inhibitors of both PKC and MEK largely prevented Prdx6 induction by KGF and, to a lesser extent, TNF-α. Interestingly, inhibition of NF-κB led to a marked increase in Prdx6 regulation in the absence or presence of inducers, suggesting a normal role for NF-κB in Prdx6 suppression. Using reporter constructs from the mouse gene, we found that the first 160 bp of the proximal promoter was sufficient for low levels of expression, and expression increased sixfold with 1200 bp of the proximal promoter. These regions were not, however, sufficient to mediate up-regulation by the known Prdx6 inducers in our system. Together, these data support multiple pathways of Prdx6 regulation and reveal important promoter regions that mediate its transcriptional regulation.

Section snippets

Cells, inducers, and inhibitors

Cells of the mouse liver hepatocyte cell line H2.35 (ATCC, Manassas, VA, USA) were cultured in tissue culture flasks in DMEM with 4% FCS and 0.2 μM dexamethasone and grown at 33°C in a 10% CO₂ atmosphere. For induction experiments, cells were cultured in the absence of serum and dexamethasone for 20–22 h, and the following concentrations of inducers were added to the culture medium for the appropriate times: 10 ng/ml KGF (Peprotech, Rocky Hill, NJ, USA), 10 ng/ml TNF-α (Alexis, Lausen,

Time course of Prdx6 induction

We first sought to determine whether each of the selected chemicals would be capable of regulating Prdx6 mRNA expression. As shown in Fig. 1A, H2.35 cells cultured under normal growing conditions express high levels of Prdx6, and these levels are significantly reduced by withdrawal of serum and dexamethasone from the culture medium for approximately 20 h. After growth arrest, we noticed strong induction of Prdx6 expression with each of the four inducers tested. In this experiment, induction was

Discussion

In the present study, we sought to investigate various inducers of growth and oxidative stress for their ability to up-regulate Prdx6 mRNA expression in H2.35 mouse liver cells and study the pathways that mediate this regulation. We determined the relative time course of Prdx6 induction in H2.35 cells in response to KGF, TNF-α, dexamethasone, and hydrogen peroxide. Expression increased within hours for each inducer, and KGF and TNF-α were the more potent inducers. Based on the diverse modes of

Acknowledgments

We thank Carolyn Stankiowicz, Michael Certo, Jaclyne Braiewa, and Christopher Caspers for preliminary investigations.

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Original ContributionInvestigating transcriptional regulation of Prdx6 in mouse liver cells

Abstract

Section snippets

Cells, inducers, and inhibitors

Time course of Prdx6 induction

Discussion

Acknowledgments

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Atherosclerosis

Am. J. Pathol.

Exp. Eye Res.

Genomics

J. Biol. Chem.

Mech. Ageing Dev.

Cytokine Growth Factor Rev.

Exp. Eye Res.

J. Biol. Chem.

J. Biol. Chem.

Gene

Biochem. Biophys. Res. Commun.

Original Contribution
Investigating transcriptional regulation of Prdx6 in mouse liver cells