Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells - PubMed (original) (raw)

Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells

Abhi K Rao et al. J Mol Endocrinol. 2009 Dec.

Abstract

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERalpha, Trx, and TrxR interact and ERalpha and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17beta-estradiol, Trx, and TrxR alter hydrogen peroxide (H(2)O(2)) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H(2)O(2) levels and transcription factor activity, aid ERalpha in regulating the expression of estrogen-responsive genes in target cells.

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Conflict of interest statement

Declaration of interest

None of the authors has a conflict of interest.

Figures

Figure 1

Trx and TrxR expression and localization. (A) The levels of Trx and TrxR were examined in nuclear extracts (10 µg) from human ERα-positive breast (MCF-7) and ERα-negative breast (MDA-MB-231), U2 osteosarcoma (U2OS), and cervical (HeLa) cancer cells. (B) Trx and TrxR expression was examined in MCF-7 cells that had been treated with ethanol (−E2) or 10 nM E2 (+E2) for 24 h. Samples were fractionated by SDS-PAGE and subjected to western analysis with a Trx- or TrxR-specific antibody. GAPDH levels were monitored to demonstrate that similar amounts of sample were loaded in each lane. (C) MCF-7 cells were treated with ethanol (−E2) or 10 nM E2 (+E2) for 24 h. Expression of Trx and TrxR was monitored using immunocyto-chemistry with Trx- and TrxR-specific antibodies. DAPI counter-staining was used to detect cell nuclei. The insert in the upper right hand corner of the −E2 images demonstrated that when the primary antibody was omitted, the secondary antibodies produced no signal. Full colour version of this figure available via

http://dx.doi.org/10.1677/JME-09-0053

Figure 2

Endogenously expressed Trx, TrxR, and ERα interact. MCF-7 cells were treated with ethanol (−E2) or 10 nM E2 (+E2) for 0.75 h and lysed. Cell extracts were incubated with (A) Trx- or (B) TrxR- specific antibody. Specifically bound proteins were eluted and subjected to western analysis with an ERα-specific antibody. MCF-7 extracts (10% input) were included in each experiment for comparison. Data shown are representative of three (A) or six (B) independent experiments.

Figure 3

E2 increases association of TrxR with endogenous, estrogen-responsive genes. MCF-7 cells were treated with ethanol (light gray bars) or 10 nM E2 for 0.75 (dark gray bars) or 24 h (black bars) and subjected to ChIP analysis with an (A) ERα-or (B) TrxR-specific antibody. Quantitative real-time PCR was used to examine the association of ERα and TrxR with the ERE-containing regions of the pS2 and PR (PR205 and PR221) genes. Data are presented as the number of copies of each estrogen-responsive gene region pulled down relative to the number of copies of 36B4 gene region pulled down (occupancy). A significant increase in the association of ERα and TrxR with these gene regions in the presence of E2 is indicated by an. asterisk (*_p_≤0.05).

Figure 4

Knocking down Trx influences estrogen responsiveness. MCF-7 cells were transfected with 50 pmol control or Trx siRNA, treated with ethanol (−E2 and light gray bars) or 10 nM E2 (+E2 and black bars) for 24 h, and processed for protein or mRNA analysis. (A) Proteins were subjected to western analysis with an antibody that recognizes Trx, PR-A and PR-B, or GAPDH. (B) RNA was isolated and cDNA was synthesized for quantitative RT-PCR analysis with primers specific to Trx, pS2, PR, cyclin G2, cyclin D1, Bcl2, ERα, and 36B4 (internal control) mRNA sequences. Data from three independent experiments, which had been performed in triplicate, were combined and are presented as the mean ±

s.e.m

. ANOVA was used to detect significant differences in mRNA levels in response to E2 (*_P_≤0.05) or in response to Trx siRNA (#_P_≤0.05).

Figure 5

Knocking down TrxR influences estrogen responsiveness. MCF-7 cells were transfected with 50 pmol control or TrxR siRNA, treated with ethanol (–E2 and light gray bars) or 10 nM E2 (+E2 and black bars) for 24 h, and processed for protein or mRNA analysis. (A) Proteins were subjected to western analysis with an antibody that recognizes TrxR, PR-A and PR-B, or GAPDH. (B) RNA was isolated and cDNA was synthesized for quantitative RT-PCR analysis with primers specific to TrxR, pS2, PR, cyclin G2, cyclin D1, Bcl2, ERα, and 36B4 (internal control) mRNA sequences. Data from three independent experiments, which had been performed in triplicate, were combined and are presented as the mean±

S.E.M

. ANOVA was used to detect significant differences in mRNA levels in response to E2 (*_P_≤0.05) or in response to TrxR siRNA (#_P_≤0.05).

Figure 6

Trx, TrxR, and E2 modulate H2O2 levels. MCF-7 cells were transfected with 50 pmol control, Trx, or TrxR siRNA and treated with ethanol (−E2) or 10 nM E2 (+E2) for 24 h. Cell extracts were prepared and incubated with Amplex Red to detect the levels of H2O2. Data from three independent experiments were combined and are expressed as the mean±

S.E.M

. ANOVA was used to detect significant differences in the levels of H2O2 in the presence of E2 (*_P_≤0.05) or in response to Trx or TrxR siRNA (#_P_≤0.05).

Figure 7

Oxidative stress response protein forms an interconnected network that alters ROS distribution and influences estrogen responsiveness. (A) Oxidized Trx (Trx-ox) is reduced (Trx-red) by TrxR using NADPH as a cofactor. SOD1 dismutates superoxide to form H2O2 and reduced Trx activates peroxiredoxins (Prx) to help eliminate H2O2. Trx, Ape1, and PDI reduce zinc finger proteins, enhance interaction with their cognate-binding sites in DNA, and alter transcription. Adapted from Webster et al. 2001. (B) Trx, TrxR, SOD1, PDI, and Ape1 form an interconnected network of proteins (Lundstrom & Holmgren 1990, Cheung et al. 1999, Wei et al. 2000, Webster et al. 2001, Atkin et al. 2006, Schultz-Norton et al. 2006, 2008, Ando et al. 2008, Rao et al. 2008, Curtis et al. 2009) that are recruited to the DNA-bound ERα (Schultz-Norton et al. 2008) and influence ERα-mediated gene expression (Schultz-Norton et al. 2006, Rao et al. 2008, Curtis et al. 2009).

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Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells - PubMed (original) (raw)