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Frequency Modulated Translocational Oscillations of Nrf2 Mediate the Antioxidant Response Element Cytoprotective Transcriptional Response.

Xue M, Momiji H, Rabbani N, Barker G, Bretschneider T, Shmygol A, Rand DA, Thornalley PJ - Antioxid. Redox Signal. (2014)

Bottom Line: Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects.We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration.We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

View Article: PubMed Central - PubMed

Affiliation: 1 Clinical Sciences Research Laboratories, Warwick Medical School, University Hospital , University of Warwick, Coventry, United Kingdom .

ABSTRACT

Aims: Stress responsive signaling coordinated by nuclear factor erythroid 2-related factor 2 (Nrf2) provides an adaptive response for protection of cells against toxic insults, oxidative stress and metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). The aim of this study was to examine how Nrf2 signals cell stress status and regulates transcription to maintain homeostasis.

Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component.

Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

Conclusion: Frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response.

No MeSH data available.


Related in: MedlinePlus

Validation of Nrf2 subcellular location by GFP-Nrf2 reporter and ARE-linked transcriptional response in human vascular endothelial HMEC-1 cellsin vitro. (A) Immunoprecipitation with anti-Nrf2 antibody (H300) and Western blot for Nrf2 with EP1808Y antibody. Lanes 1–3, untransfected cells; lanes 4 and 5, cells transfected with pcDNA3-EGFP-C4-Nrf2 expressing GFP-Nrf2. Nrf2 and GFP-Nrf2 bands were at 98 and 125 kDa, respectively. (B–E) Fluorescence microscopy of HMEC-1 cells for detection of Nrf2 and GFP by immunofluorescence. Two cells in the field of view have Nrf2 mostly in the cytoplasm (left) and in the cytoplasm and nucleus (right). (B) Nuclear staining with DAPI, (C) Immunofluorescence for Nrf2 with EP1808Y primary antibody and Alexa Fluor®488 conjugated goat anti-rabbit IgG secondary antibody, (D) Immunofluorescence for GFP with GFP-1020 primary antibody and Alexa Fluor®555 conjugated goat anti-chicken IgG secondary antibody, and (E) overlay image. (F–Q). Changes in gene expression, mRNA levels normalized to housekeeping genes in control (open square, dashed line) and SFN stimulated cells (filled square, solid line). (F) quinone reductase, NQO1, (G) glutathione reductase GSR, (H) γ-glutamylcysteine ligase (regulatory subunit) GCLM, (I) thioredoxin reductase TXNRD1, (J) ferritin FTH1, (K) heme oxygenase HMOX1, (L) glucose-6-phosphate dehydrogenase G6PDH, (M) transketolase TKT, (N) sequestosome-1/p62 SQSTM1 and (O, P, and Q), aldoketo reductase isoforms AK1B1,AK1C1 and AK1C3. Key: dashed line with open squares, control; solid line with filled squares, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Significance: all control and SFN time courses were significantly different in repeated measures analysis and at individual time points (p<0.001, t-test) except for HMOX1 from 24 to 72 h and SQSTM1 at 48 h. Indicators of significance are omitted for clarity. Stimulation of HMEC-1 cells with SFN produced increased expression of ARE-linked genes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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f3: Validation of Nrf2 subcellular location by GFP-Nrf2 reporter and ARE-linked transcriptional response in human vascular endothelial HMEC-1 cellsin vitro. (A) Immunoprecipitation with anti-Nrf2 antibody (H300) and Western blot for Nrf2 with EP1808Y antibody. Lanes 1–3, untransfected cells; lanes 4 and 5, cells transfected with pcDNA3-EGFP-C4-Nrf2 expressing GFP-Nrf2. Nrf2 and GFP-Nrf2 bands were at 98 and 125 kDa, respectively. (B–E) Fluorescence microscopy of HMEC-1 cells for detection of Nrf2 and GFP by immunofluorescence. Two cells in the field of view have Nrf2 mostly in the cytoplasm (left) and in the cytoplasm and nucleus (right). (B) Nuclear staining with DAPI, (C) Immunofluorescence for Nrf2 with EP1808Y primary antibody and Alexa Fluor®488 conjugated goat anti-rabbit IgG secondary antibody, (D) Immunofluorescence for GFP with GFP-1020 primary antibody and Alexa Fluor®555 conjugated goat anti-chicken IgG secondary antibody, and (E) overlay image. (F–Q). Changes in gene expression, mRNA levels normalized to housekeeping genes in control (open square, dashed line) and SFN stimulated cells (filled square, solid line). (F) quinone reductase, NQO1, (G) glutathione reductase GSR, (H) γ-glutamylcysteine ligase (regulatory subunit) GCLM, (I) thioredoxin reductase TXNRD1, (J) ferritin FTH1, (K) heme oxygenase HMOX1, (L) glucose-6-phosphate dehydrogenase G6PDH, (M) transketolase TKT, (N) sequestosome-1/p62 SQSTM1 and (O, P, and Q), aldoketo reductase isoforms AK1B1,AK1C1 and AK1C3. Key: dashed line with open squares, control; solid line with filled squares, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Significance: all control and SFN time courses were significantly different in repeated measures analysis and at individual time points (p<0.001, t-test) except for HMOX1 from 24 to 72 h and SQSTM1 at 48 h. Indicators of significance are omitted for clarity. Stimulation of HMEC-1 cells with SFN produced increased expression of ARE-linked genes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Mentions: We also made test transfections to determine the extent to which Nrf2-GFP increases the total cellular content of Nrf2 and to check that GFP-Nrf2 faithfully reports the cellular location of Nrf2. Transfection with the vector pcDNA3-EGFP-C4-Nrf2 produces expression of Nrf2-GFP comprising the human Nrf2 domain (sequence mass 68 kDa, with N-terminal tag of GFP, 27 kDa sequence mass). Antibodies raised to the C-terminal domain of Nrf2, EP1808Y, detected both Nrf2 and Nrf2-GFP with equal affinity. Western blotting with EP1808Y antibody detected a band at 98 kDa from control, untransfected HMEC-1 cells (consistent with electrophoretic mobility of Nrf2) (32, 65), and bands at 98 and 125 kDa from transfected HMEC-1 (consistent with electrophoretic mobility of Nrf2 and Nrf2-GFP) (Fig. 3A). Densitometry of these bands produced an intensity ratio of 100:4. Immunoblotting of Nrf2 with H300 antibodies raised to an Nrf2 N-terminal epitope gave similar results—intensity ratio of 100:7, indicating that expression of GFP-Nrf2 produced only a minor increase in the total Nrf2 pool of 4%–7%. Immunofluorescence micrographs of transfected HMEC-1 cells with anti-Nrf2 antibody produced staining for some cells mostly in the cytosol, others mostly nucleus or both cytosol and nucleus. Irrespective of where anti-Nrf2 immunostaining was located (green), immunofluorescence of GFP (red) showed co-localization with Nrf2 (Fig. 3B–E). Addition of stimulants did not change total GFP-Nrf2 fluorescence. Moreover, studies with double transfection of cells to express GFP-Nrf2 and destabilized Venus-ARE-linked transcription reporter with monitoring of Nrf2 translocation and ARE-linked transcription before and after SFN stimulation indicated that cells with increased Nrf2 translocation frequency were those with increased ARE transcription activity.


Frequency Modulated Translocational Oscillations of Nrf2 Mediate the Antioxidant Response Element Cytoprotective Transcriptional Response.

Xue M, Momiji H, Rabbani N, Barker G, Bretschneider T, Shmygol A, Rand DA, Thornalley PJ - Antioxid. Redox Signal. (2014)

Validation of Nrf2 subcellular location by GFP-Nrf2 reporter and ARE-linked transcriptional response in human vascular endothelial HMEC-1 cellsin vitro. (A) Immunoprecipitation with anti-Nrf2 antibody (H300) and Western blot for Nrf2 with EP1808Y antibody. Lanes 1–3, untransfected cells; lanes 4 and 5, cells transfected with pcDNA3-EGFP-C4-Nrf2 expressing GFP-Nrf2. Nrf2 and GFP-Nrf2 bands were at 98 and 125 kDa, respectively. (B–E) Fluorescence microscopy of HMEC-1 cells for detection of Nrf2 and GFP by immunofluorescence. Two cells in the field of view have Nrf2 mostly in the cytoplasm (left) and in the cytoplasm and nucleus (right). (B) Nuclear staining with DAPI, (C) Immunofluorescence for Nrf2 with EP1808Y primary antibody and Alexa Fluor®488 conjugated goat anti-rabbit IgG secondary antibody, (D) Immunofluorescence for GFP with GFP-1020 primary antibody and Alexa Fluor®555 conjugated goat anti-chicken IgG secondary antibody, and (E) overlay image. (F–Q). Changes in gene expression, mRNA levels normalized to housekeeping genes in control (open square, dashed line) and SFN stimulated cells (filled square, solid line). (F) quinone reductase, NQO1, (G) glutathione reductase GSR, (H) γ-glutamylcysteine ligase (regulatory subunit) GCLM, (I) thioredoxin reductase TXNRD1, (J) ferritin FTH1, (K) heme oxygenase HMOX1, (L) glucose-6-phosphate dehydrogenase G6PDH, (M) transketolase TKT, (N) sequestosome-1/p62 SQSTM1 and (O, P, and Q), aldoketo reductase isoforms AK1B1,AK1C1 and AK1C3. Key: dashed line with open squares, control; solid line with filled squares, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Significance: all control and SFN time courses were significantly different in repeated measures analysis and at individual time points (p<0.001, t-test) except for HMOX1 from 24 to 72 h and SQSTM1 at 48 h. Indicators of significance are omitted for clarity. Stimulation of HMEC-1 cells with SFN produced increased expression of ARE-linked genes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4556091&req=5

f3: Validation of Nrf2 subcellular location by GFP-Nrf2 reporter and ARE-linked transcriptional response in human vascular endothelial HMEC-1 cellsin vitro. (A) Immunoprecipitation with anti-Nrf2 antibody (H300) and Western blot for Nrf2 with EP1808Y antibody. Lanes 1–3, untransfected cells; lanes 4 and 5, cells transfected with pcDNA3-EGFP-C4-Nrf2 expressing GFP-Nrf2. Nrf2 and GFP-Nrf2 bands were at 98 and 125 kDa, respectively. (B–E) Fluorescence microscopy of HMEC-1 cells for detection of Nrf2 and GFP by immunofluorescence. Two cells in the field of view have Nrf2 mostly in the cytoplasm (left) and in the cytoplasm and nucleus (right). (B) Nuclear staining with DAPI, (C) Immunofluorescence for Nrf2 with EP1808Y primary antibody and Alexa Fluor®488 conjugated goat anti-rabbit IgG secondary antibody, (D) Immunofluorescence for GFP with GFP-1020 primary antibody and Alexa Fluor®555 conjugated goat anti-chicken IgG secondary antibody, and (E) overlay image. (F–Q). Changes in gene expression, mRNA levels normalized to housekeeping genes in control (open square, dashed line) and SFN stimulated cells (filled square, solid line). (F) quinone reductase, NQO1, (G) glutathione reductase GSR, (H) γ-glutamylcysteine ligase (regulatory subunit) GCLM, (I) thioredoxin reductase TXNRD1, (J) ferritin FTH1, (K) heme oxygenase HMOX1, (L) glucose-6-phosphate dehydrogenase G6PDH, (M) transketolase TKT, (N) sequestosome-1/p62 SQSTM1 and (O, P, and Q), aldoketo reductase isoforms AK1B1,AK1C1 and AK1C3. Key: dashed line with open squares, control; solid line with filled squares, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Significance: all control and SFN time courses were significantly different in repeated measures analysis and at individual time points (p<0.001, t-test) except for HMOX1 from 24 to 72 h and SQSTM1 at 48 h. Indicators of significance are omitted for clarity. Stimulation of HMEC-1 cells with SFN produced increased expression of ARE-linked genes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
Mentions: We also made test transfections to determine the extent to which Nrf2-GFP increases the total cellular content of Nrf2 and to check that GFP-Nrf2 faithfully reports the cellular location of Nrf2. Transfection with the vector pcDNA3-EGFP-C4-Nrf2 produces expression of Nrf2-GFP comprising the human Nrf2 domain (sequence mass 68 kDa, with N-terminal tag of GFP, 27 kDa sequence mass). Antibodies raised to the C-terminal domain of Nrf2, EP1808Y, detected both Nrf2 and Nrf2-GFP with equal affinity. Western blotting with EP1808Y antibody detected a band at 98 kDa from control, untransfected HMEC-1 cells (consistent with electrophoretic mobility of Nrf2) (32, 65), and bands at 98 and 125 kDa from transfected HMEC-1 (consistent with electrophoretic mobility of Nrf2 and Nrf2-GFP) (Fig. 3A). Densitometry of these bands produced an intensity ratio of 100:4. Immunoblotting of Nrf2 with H300 antibodies raised to an Nrf2 N-terminal epitope gave similar results—intensity ratio of 100:7, indicating that expression of GFP-Nrf2 produced only a minor increase in the total Nrf2 pool of 4%–7%. Immunofluorescence micrographs of transfected HMEC-1 cells with anti-Nrf2 antibody produced staining for some cells mostly in the cytosol, others mostly nucleus or both cytosol and nucleus. Irrespective of where anti-Nrf2 immunostaining was located (green), immunofluorescence of GFP (red) showed co-localization with Nrf2 (Fig. 3B–E). Addition of stimulants did not change total GFP-Nrf2 fluorescence. Moreover, studies with double transfection of cells to express GFP-Nrf2 and destabilized Venus-ARE-linked transcription reporter with monitoring of Nrf2 translocation and ARE-linked transcription before and after SFN stimulation indicated that cells with increased Nrf2 translocation frequency were those with increased ARE transcription activity.

Bottom Line: Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects.We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration.We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

View Article: PubMed Central - PubMed

Affiliation: 1 Clinical Sciences Research Laboratories, Warwick Medical School, University Hospital , University of Warwick, Coventry, United Kingdom .

ABSTRACT

Aims: Stress responsive signaling coordinated by nuclear factor erythroid 2-related factor 2 (Nrf2) provides an adaptive response for protection of cells against toxic insults, oxidative stress and metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). The aim of this study was to examine how Nrf2 signals cell stress status and regulates transcription to maintain homeostasis.

Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component.

Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

Conclusion: Frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response.

No MeSH data available.


Related in: MedlinePlus