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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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Differential regulation of NRF2 and BACH1 activation. Immunoblots illustrating differential expression of NRF2 and BACH1 protein following treatment of HaCaT cells with 25 μM arsenite, 5 μM MG132, 25 μM hemin or MG132 + hemin combined. HaCaT cells were treated as indicated for 3 h or co-treated with 5 μM cycloheximide (CHX) following a 30-min CHX pretreatment. (A) Total cellular proteins (20 μg) from whole cell lysates. (B) Proteins extracts (10 μg) from nuclear lysates. (C) Proteins extracts (20 μg) from cytosolic lysates. Blots are representative of 2–3 separate experiments.
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Figure 3: Differential regulation of NRF2 and BACH1 activation. Immunoblots illustrating differential expression of NRF2 and BACH1 protein following treatment of HaCaT cells with 25 μM arsenite, 5 μM MG132, 25 μM hemin or MG132 + hemin combined. HaCaT cells were treated as indicated for 3 h or co-treated with 5 μM cycloheximide (CHX) following a 30-min CHX pretreatment. (A) Total cellular proteins (20 μg) from whole cell lysates. (B) Proteins extracts (10 μg) from nuclear lysates. (C) Proteins extracts (20 μg) from cytosolic lysates. Blots are representative of 2–3 separate experiments.

Mentions: To investigate the relative contributions of BACH1 and NRF2 to HMOX1 expression, we differentially regulated their activities by treating cells with the proteasome inhibitor MG132 or with hemin. MG132 indirectly causes NRF2 activation by inhibiting KEAP1-dependent proteasomal degradation (26). Hemin inactivates BACH1 through interaction with its multiple heme-binding motifs leading to a conformational change and nuclear export (27,28). Treatment of HaCaT cells with MG132 for 3 h induces extensive NRF2 accumulation in whole cell lysates relative to untreated control cells (Figure 3A). This increase in NRF2 is almost entirely localized to the nuclear fraction (Figure 3B) with negligible levels detected in the cytosolic fraction (Figure 3C). In contrast to NRF2, BACH1 levels are only slightly affected by MG132 treatment in whole cell lysates or nuclear distribution (Figure 3). Hemin treatment contrasts with MG132 treatment in that it prominently triggers BACH1 inactivation, which manifests as a significant decrease in whole cell BACH1 protein levels (Figure 3A) and as its disappearance from the nuclear fraction (Figure 3B). Interestingly, this effect is not associated so much with nuclear efflux to the cytosolic compartment as occurs following arsenite treatment, but rather is associated with a net loss of total BACH1 (Figure 3A). Co-treatment of MG132 and hemin blocks this net loss of BACH1 (Figure 3A) and results in its cytoplasmic accumulation due to efflux from the nucleus (Figure 3C). Thus, inactivation of BACH1 by hemin triggers both its nuclear export and subsequent proteasomal degradation. In contrast to hemin, arsenite only mediates subcellular redistribution of BACH1, as indicated by nuclear efflux, without notable protein loss in whole cell lysates (Figure 3). Importantly, hemin treatment has only minimal effects on NRF2 activation, as indicated by the relative absence of NRF2 accumulation in either whole cell extracts or nuclear fractions. Co-treatment with hemin plus MG132 produces a combined pattern of NRF2 nuclear translocation and BACH1 efflux similar to that elicited by arsenite. Consistent with these findings, the expression of HMOX1 protein (Figure 3A) is associated only with treatments that result in BACH1 efflux from the nucleus, but not with NRF2 activation alone.Figure 3.


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

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

Differential regulation of NRF2 and BACH1 activation. Immunoblots illustrating differential expression of NRF2 and BACH1 protein following treatment of HaCaT cells with 25 μM arsenite, 5 μM MG132, 25 μM hemin or MG132 + hemin combined. HaCaT cells were treated as indicated for 3 h or co-treated with 5 μM cycloheximide (CHX) following a 30-min CHX pretreatment. (A) Total cellular proteins (20 μg) from whole cell lysates. (B) Proteins extracts (10 μg) from nuclear lysates. (C) Proteins extracts (20 μg) from cytosolic lysates. Blots are representative of 2–3 separate experiments.
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Figure 3: Differential regulation of NRF2 and BACH1 activation. Immunoblots illustrating differential expression of NRF2 and BACH1 protein following treatment of HaCaT cells with 25 μM arsenite, 5 μM MG132, 25 μM hemin or MG132 + hemin combined. HaCaT cells were treated as indicated for 3 h or co-treated with 5 μM cycloheximide (CHX) following a 30-min CHX pretreatment. (A) Total cellular proteins (20 μg) from whole cell lysates. (B) Proteins extracts (10 μg) from nuclear lysates. (C) Proteins extracts (20 μg) from cytosolic lysates. Blots are representative of 2–3 separate experiments.
Mentions: To investigate the relative contributions of BACH1 and NRF2 to HMOX1 expression, we differentially regulated their activities by treating cells with the proteasome inhibitor MG132 or with hemin. MG132 indirectly causes NRF2 activation by inhibiting KEAP1-dependent proteasomal degradation (26). Hemin inactivates BACH1 through interaction with its multiple heme-binding motifs leading to a conformational change and nuclear export (27,28). Treatment of HaCaT cells with MG132 for 3 h induces extensive NRF2 accumulation in whole cell lysates relative to untreated control cells (Figure 3A). This increase in NRF2 is almost entirely localized to the nuclear fraction (Figure 3B) with negligible levels detected in the cytosolic fraction (Figure 3C). In contrast to NRF2, BACH1 levels are only slightly affected by MG132 treatment in whole cell lysates or nuclear distribution (Figure 3). Hemin treatment contrasts with MG132 treatment in that it prominently triggers BACH1 inactivation, which manifests as a significant decrease in whole cell BACH1 protein levels (Figure 3A) and as its disappearance from the nuclear fraction (Figure 3B). Interestingly, this effect is not associated so much with nuclear efflux to the cytosolic compartment as occurs following arsenite treatment, but rather is associated with a net loss of total BACH1 (Figure 3A). Co-treatment of MG132 and hemin blocks this net loss of BACH1 (Figure 3A) and results in its cytoplasmic accumulation due to efflux from the nucleus (Figure 3C). Thus, inactivation of BACH1 by hemin triggers both its nuclear export and subsequent proteasomal degradation. In contrast to hemin, arsenite only mediates subcellular redistribution of BACH1, as indicated by nuclear efflux, without notable protein loss in whole cell lysates (Figure 3). Importantly, hemin treatment has only minimal effects on NRF2 activation, as indicated by the relative absence of NRF2 accumulation in either whole cell extracts or nuclear fractions. Co-treatment with hemin plus MG132 produces a combined pattern of NRF2 nuclear translocation and BACH1 efflux similar to that elicited by arsenite. Consistent with these findings, the expression of HMOX1 protein (Figure 3A) is associated only with treatments that result in BACH1 efflux from the nucleus, but not with NRF2 activation alone.Figure 3.

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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