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Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1.

Reichard JF, Motz GT, Puga A - Nucleic Acids Res. (2007)

Bottom Line: In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1.By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction.Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, Cincinnati, OH 45267-0056, USA. john.reichard@childrens.harvard.edu

ABSTRACT
Oxidative stress activates the transcription factor NRF2, which in turn binds cis-acting antioxidant response element (ARE) enhancers and induces expression of protective antioxidant genes. In contrast, the transcriptional repressor BACH1 binds ARE-like enhancers in cells naïve to oxidative stress and antagonizes NRF2 binding until it becomes inactivated by pro-oxidants. Here, we describe the dynamic roles of BACH1 and NRF2 in the transcription of the heme oxygenase-1 (HMOX1) gene. HMOX1 induction, elicited by arsenite-mediated oxidative stress, follows inactivation of BACH1 and precedes activation of NRF2. BACH1 repression is dominant over NRF2-mediated HMOX1 transcription and inactivation of BACH1 is a prerequisite for HMOX1 induction. In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1. By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction. Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

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Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 μg) illustrating the effect of 25 μM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in expression of NRF2 and BACH1 proteins normalized to β-actin levels. (C) Representative immunoblots of nuclear (10 μg) and cytosolic proteins (20 μg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 μM). (D) Graphical representation of three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to β-actin levels. The presence of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean ± SEM of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 μM arsenite. HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to β-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified in triplicate.
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Figure 1: Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 μg) illustrating the effect of 25 μM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in expression of NRF2 and BACH1 proteins normalized to β-actin levels. (C) Representative immunoblots of nuclear (10 μg) and cytosolic proteins (20 μg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 μM). (D) Graphical representation of three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to β-actin levels. The presence of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean ± SEM of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 μM arsenite. HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to β-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified in triplicate.

Mentions: To characterize activation of the antioxidant response following arsenite exposure, we first characterized cellular and subcellular changes in NRF2 and BACH1 disposition. In control cells, NRF2 is present at very low levels in whole cell extracts attributable to ongoing proteasomal degradation mediated by KEAP1 (25). After treatment with 25 μM arsenite, NRF2 accumulates to high levels in whole cell extracts consistent with oxidative inactivation of KEAP1 (23) (Figure 1A and B). Importantly, NRF2 accumulation is negligible during the first hour after arsenite treatment, and does not reach maximum levels until 3 h after treatment. This initial lag period represents the time necessary for translational synthesis of new NRF2 protein (26), which precedes nuclear translocation and subsequent ARE-mediated gene induction. In comparison to NRF2, BACH1 gradually accumulates in whole cell extracts over 5 h after treatment. The extent to which BACH1 accumulation is attributable to protein stabilization is unknown; however, no change in BACH1 mRNA expression was observed (data not shown).Figure 1.


Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1.

Reichard JF, Motz GT, Puga A - Nucleic Acids Res. (2007)

Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 μg) illustrating the effect of 25 μM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in expression of NRF2 and BACH1 proteins normalized to β-actin levels. (C) Representative immunoblots of nuclear (10 μg) and cytosolic proteins (20 μg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 μM). (D) Graphical representation of three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to β-actin levels. The presence of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean ± SEM of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 μM arsenite. HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to β-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified in triplicate.
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Figure 1: Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 μg) illustrating the effect of 25 μM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in expression of NRF2 and BACH1 proteins normalized to β-actin levels. (C) Representative immunoblots of nuclear (10 μg) and cytosolic proteins (20 μg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 μM). (D) Graphical representation of three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to β-actin levels. The presence of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean ± SEM of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 μM arsenite. HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to β-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified in triplicate.
Mentions: To characterize activation of the antioxidant response following arsenite exposure, we first characterized cellular and subcellular changes in NRF2 and BACH1 disposition. In control cells, NRF2 is present at very low levels in whole cell extracts attributable to ongoing proteasomal degradation mediated by KEAP1 (25). After treatment with 25 μM arsenite, NRF2 accumulates to high levels in whole cell extracts consistent with oxidative inactivation of KEAP1 (23) (Figure 1A and B). Importantly, NRF2 accumulation is negligible during the first hour after arsenite treatment, and does not reach maximum levels until 3 h after treatment. This initial lag period represents the time necessary for translational synthesis of new NRF2 protein (26), which precedes nuclear translocation and subsequent ARE-mediated gene induction. In comparison to NRF2, BACH1 gradually accumulates in whole cell extracts over 5 h after treatment. The extent to which BACH1 accumulation is attributable to protein stabilization is unknown; however, no change in BACH1 mRNA expression was observed (data not shown).Figure 1.

Bottom Line: In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1.By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction.Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, Cincinnati, OH 45267-0056, USA. john.reichard@childrens.harvard.edu

ABSTRACT
Oxidative stress activates the transcription factor NRF2, which in turn binds cis-acting antioxidant response element (ARE) enhancers and induces expression of protective antioxidant genes. In contrast, the transcriptional repressor BACH1 binds ARE-like enhancers in cells naïve to oxidative stress and antagonizes NRF2 binding until it becomes inactivated by pro-oxidants. Here, we describe the dynamic roles of BACH1 and NRF2 in the transcription of the heme oxygenase-1 (HMOX1) gene. HMOX1 induction, elicited by arsenite-mediated oxidative stress, follows inactivation of BACH1 and precedes activation of NRF2. BACH1 repression is dominant over NRF2-mediated HMOX1 transcription and inactivation of BACH1 is a prerequisite for HMOX1 induction. In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1. By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction. Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

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Related in: MedlinePlus