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AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1.

ABSTRACT: 5'-AMP-activated protein kinase (AMPK) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) are main players in the cellular adaptive response to metabolic and oxidative/xenobiotic stress, respectively. AMPK does not only balance the rate of fuel catabolism versus anabolism but also emerges as regulator of gene expression. We here examined the influence of AMPK on Nrf2-dependent gene transcription and the potential interplay of the two cellular stress hubs. Using gene expression analyses in wt and AMPK?1 -/- or Nrf2 -/- mouse embryonal fibroblasts, we could show that AMPK only affected a portion of the entire of Nrf2-dependent transcriptome upon exposure to the Nrf2 activator sulforaphane (Sfn). Focusing on selected genes with positive regulation by Nrf2 and either positive or no further regulation by AMPK, we revealed that altered Nrf2 levels could not account for the distinct extent of transactivation of certain Nrf2 targets in wt and AMPK -/- cells (assessed by immunoblot). FAIRE-qPCR largely excluded distinct chromatin accessibility of selected Nrf2-responsive antioxidant response elements (ARE) within the regulatory gene regions in wt and AMPK-/- cells. However, expression analyses and ChIP-qPCR showed that in AMPK-/- cells, levels of BTB and CNC homology 1 (Bach1), a competitor of Nrf2 for ARE sites with predominant repressor function, were higher, and Bach1 also bound to a greater relative extent to the examined ARE sites when compared to Nrf2. The negative influence of AMPK on Bach1 was confirmed by pharmacological and genetic approaches and occurred at the level of mRNA synthesis. Overall, the observed AMPK-mediated boost in transactivation of a subset of Nrf2 target genes involves downregulation of Bach1 and subsequent favored binding of activating Nrf2 over repressing Bach1 to the examined ARE sites.

SUBMITTER: Fischhuber K

PROVIDER: S-EPMC7372114 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1.

Fischhuber Katrin K Matzinger Manuel M Heiss Elke H EH

Frontiers in cell and developmental biology 20200714

5'-AMP-activated protein kinase (AMPK) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) are main players in the cellular adaptive response to metabolic and oxidative/xenobiotic stress, respectively. AMPK does not only balance the rate of fuel catabolism versus anabolism but also emerges as regulator of gene expression. We here examined the influence of AMPK on Nrf2-dependent gene transcription and the potential interplay of the two cellular stress hubs. Using gene ...[more]

PMID: 32760724

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Project description:AMP-activated protein kinase (AMPK) enhances glucose transporter GLUT4 regulation. AMPK also suppresses energy-consuming pathways such as cholesterol synthesis. Interestingly, recent in vitro and in vivo data suggest that excess membrane cholesterol impairs GLUT4 regulation. Therefore, this study tested whether a beneficial, GLUT4-regulatory aspect of AMPK stimulation involved cholesterol lowering. Using L6 myotubes stably expressing an exofacial myc-epitope-tagged-GLUT4, AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-?-d-ribonucleoside (AICAR; 45 min, 1 mm) or 2,4-dinitrophenol (DNP; 30 min, 200 ?m) increased cell surface GLUT4myc labeling by approximately ? 25% (P < 0.05). Insulin (20 min, 100 nm) also increased GLUT4myc labeling by about 50% (P < 0.05), which was further enhanced (? 25%, P < 0.05) by AICAR or DNP. Consistent with AMPK-mediated suppression of cholesterol synthesis, AICAR and DNP decreased membrane cholesterol by 20-25% (P < 0.05). Whereas AMPK knockdown prevented the enhanced basal and insulin-stimulated GLUT4myc labeling by AICAR and DNP, cholesterol replenishment only blocked the AMPK-associated enhancement in insulin action. Cells cultured in a hyperinsulinemic milieu, resembling conditions in vivo that promote the progression/worsening of insulin resistance, displayed an increase in membrane cholesterol. This occurred concomitantly with a loss of cortical filamentous actin (F-actin) and defects in GLUT4 regulation by insulin. These derangements were prevented by AMPK stimulation. Examination of skeletal muscle from insulin-resistant Zucker rats revealed a similar elevation in membrane cholesterol and loss of F-actin. Lowering cholesterol to control levels restored F-actin structure and insulin sensitivity. In conclusion, these data suggest a novel aspect of GLUT4 regulation by AMPK involves membrane cholesterol lowering. Moreover, this AMPK-mediated process protected against hyperinsulinemia-induced insulin resistance.

Project description:BackgroundMacrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Heme oxygenase 1 degrades the heme moiety and releases inorganic iron that is stored in ferritin or exported to the plasma via the iron export protein ferroportin. In the plasma, iron binds to transferrin and is made available for de novo red cell synthesis. The aim of this study was to gain insight into the regulatory mechanisms that control the transcriptional response of iron export protein ferroportin to hemoglobin in macrophages.Design and methodsIron export protein ferroportin mRNA expression was analyzed in RAW264.7 mouse macrophages in response to hemoglobin, heme, ferric ammonium citrate or protoporphyrin treatment or to siRNA mediated knockdown or overexpression of Btb And Cnc Homology 1 or nuclear accumulation of Nuclear Factor Erythroid 2-like. Iron export protein ferroportin promoter activity was analyzed using reporter constructs that contain specific truncations of the iron export protein ferroportin promoter or mutations in a newly identified MARE/ARE element.ResultsWe show that iron export protein ferroportin is transcriptionally co-regulated with heme oxygenase 1 by heme, a degradation product of hemoglobin. The protoporphyrin ring of heme is sufficient to increase iron export protein ferroportin transcriptional activity while the iron released from the heme moiety controls iron export protein ferroportin translation involving the IRE in the 5'untranslated region. Transcription of iron export protein ferroportin is inhibited by Btb and Cnc Homology 1 and activated by Nuclear Factor Erythroid 2-like involving a MARE/ARE element located at position -7007/-7016 of the iron export protein ferroportin promoter.ConclusionsThis finding suggests that heme controls a macrophage iron recycling regulon involving Btb and Cnc Homology 1 and Nuclear Factor Erythroid 2-like to assure the coordinated degradation of heme by heme oxygenase 1, iron storage and detoxification by ferritin, and iron export by iron export protein ferroportin.

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AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1.

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AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1.

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