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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

Trapping of Nrf2 in the nucleus and Nrf2 acetylation block Nrf2 transcriptional activity. (A) Fluorescence microscopy image frames of HMEC-1 cells transfected to express GFP-Nrf2 incubated with LMB for 2 h and cycloheximide (10 μg/ml) added. Frame time (left to right): 0, 40, 80 and 120 min. (B, C) Effect of LMB (2 ng/ml) on NQO1-ARE transcriptional activity in the absence and presence of SFN and QTN, respectively. Cells were preincubated for 2 h with LMB and then 6 h with and without Nrf2 activator. (D) Effect of TAS (150 and 300 nM) on NQO1-ARE transcriptional activity in the absence and presence of SFN (mean±SD, n=3). LMB, leptomycin B; TAS, Trichostatin A. Significance: (A, C–F, H, I), **p<0.01 and ***p<0.001 with respect to unstimulated control; and ○○○p<0.001 with respect to SFN (B, D) or QTN (C) stimulated controls (t-test).
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f7: Trapping of Nrf2 in the nucleus and Nrf2 acetylation block Nrf2 transcriptional activity. (A) Fluorescence microscopy image frames of HMEC-1 cells transfected to express GFP-Nrf2 incubated with LMB for 2 h and cycloheximide (10 μg/ml) added. Frame time (left to right): 0, 40, 80 and 120 min. (B, C) Effect of LMB (2 ng/ml) on NQO1-ARE transcriptional activity in the absence and presence of SFN and QTN, respectively. Cells were preincubated for 2 h with LMB and then 6 h with and without Nrf2 activator. (D) Effect of TAS (150 and 300 nM) on NQO1-ARE transcriptional activity in the absence and presence of SFN (mean±SD, n=3). LMB, leptomycin B; TAS, Trichostatin A. Significance: (A, C–F, H, I), **p<0.01 and ***p<0.001 with respect to unstimulated control; and ○○○p<0.001 with respect to SFN (B, D) or QTN (C) stimulated controls (t-test).

Mentions: To test the transactivational activity of Nrf2 with prolonged dwell time in the nucleus we trapped Nrf2 in the nucleus by addition of leptomycin B (LMB)—a highly specific and potent inhibitor of crm1 (29) used previously in mechanistic studies of Nrf2 nuclear export (25). Addition of LMB to HMEC-1 cells transfected to express GFP-Nrf2 showed GFP-Nrf2 fluorescence localized in the cell nucleus. Furthermore, with concurrent block of de novo Nrf2 synthesis by addition of cycloheximide, Nrf2 protein was degraded in the nucleus with a half-life of 66 (48–195) min (n=8) (Fig. 7A). A recent estimate of total cellular half-life of Nrf2 was 62 min (48). Nuclear proteolysis of Nrf2, reported previously (18), suggests that Nrf2 is not being spared from degradation even when free of Keap1 and located in the nucleus. This also confirmed that Nrf2 is exported from the nucleus via crm1 and may be degraded within the nucleus. Addition of LMB produced inhibition of constitutive NQO1-ARE transcriptional response and SFN and QTN-induced NQO1-ARE transcriptional response (Fig. 7B, C). Our regulatory model invokes nuclear inactivation of Nrf2, mediated by acetylation as found previously (40). Nrf2 is an established target for reversible acetylation where acetylation is removed by histone deacetylases (46). The deacetylase inhibitor Trichostatin A (TAS) markedly increases acetylation of endothelial cell proteins in the concentration range 100 nM–1 μM (49). Addition of TAS (150 and 300 nM) to HMEC-1 cells inhibited constitutive and SFN-induced ARE-linked transcriptional response (Fig. 7D), consistent with acetylation as a likely cause of inactivation of nucleus trapped Nrf2. These studies indicate that Nrf2 loses its transactivational activity with prolonged dwell time in the nucleus where Nrf2 acetylation and nuclear proteasomal degradation are involved.


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)

Trapping of Nrf2 in the nucleus and Nrf2 acetylation block Nrf2 transcriptional activity. (A) Fluorescence microscopy image frames of HMEC-1 cells transfected to express GFP-Nrf2 incubated with LMB for 2 h and cycloheximide (10 μg/ml) added. Frame time (left to right): 0, 40, 80 and 120 min. (B, C) Effect of LMB (2 ng/ml) on NQO1-ARE transcriptional activity in the absence and presence of SFN and QTN, respectively. Cells were preincubated for 2 h with LMB and then 6 h with and without Nrf2 activator. (D) Effect of TAS (150 and 300 nM) on NQO1-ARE transcriptional activity in the absence and presence of SFN (mean±SD, n=3). LMB, leptomycin B; TAS, Trichostatin A. Significance: (A, C–F, H, I), **p<0.01 and ***p<0.001 with respect to unstimulated control; and ○○○p<0.001 with respect to SFN (B, D) or QTN (C) stimulated controls (t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556091&req=5

f7: Trapping of Nrf2 in the nucleus and Nrf2 acetylation block Nrf2 transcriptional activity. (A) Fluorescence microscopy image frames of HMEC-1 cells transfected to express GFP-Nrf2 incubated with LMB for 2 h and cycloheximide (10 μg/ml) added. Frame time (left to right): 0, 40, 80 and 120 min. (B, C) Effect of LMB (2 ng/ml) on NQO1-ARE transcriptional activity in the absence and presence of SFN and QTN, respectively. Cells were preincubated for 2 h with LMB and then 6 h with and without Nrf2 activator. (D) Effect of TAS (150 and 300 nM) on NQO1-ARE transcriptional activity in the absence and presence of SFN (mean±SD, n=3). LMB, leptomycin B; TAS, Trichostatin A. Significance: (A, C–F, H, I), **p<0.01 and ***p<0.001 with respect to unstimulated control; and ○○○p<0.001 with respect to SFN (B, D) or QTN (C) stimulated controls (t-test).
Mentions: To test the transactivational activity of Nrf2 with prolonged dwell time in the nucleus we trapped Nrf2 in the nucleus by addition of leptomycin B (LMB)—a highly specific and potent inhibitor of crm1 (29) used previously in mechanistic studies of Nrf2 nuclear export (25). Addition of LMB to HMEC-1 cells transfected to express GFP-Nrf2 showed GFP-Nrf2 fluorescence localized in the cell nucleus. Furthermore, with concurrent block of de novo Nrf2 synthesis by addition of cycloheximide, Nrf2 protein was degraded in the nucleus with a half-life of 66 (48–195) min (n=8) (Fig. 7A). A recent estimate of total cellular half-life of Nrf2 was 62 min (48). Nuclear proteolysis of Nrf2, reported previously (18), suggests that Nrf2 is not being spared from degradation even when free of Keap1 and located in the nucleus. This also confirmed that Nrf2 is exported from the nucleus via crm1 and may be degraded within the nucleus. Addition of LMB produced inhibition of constitutive NQO1-ARE transcriptional response and SFN and QTN-induced NQO1-ARE transcriptional response (Fig. 7B, C). Our regulatory model invokes nuclear inactivation of Nrf2, mediated by acetylation as found previously (40). Nrf2 is an established target for reversible acetylation where acetylation is removed by histone deacetylases (46). The deacetylase inhibitor Trichostatin A (TAS) markedly increases acetylation of endothelial cell proteins in the concentration range 100 nM–1 μM (49). Addition of TAS (150 and 300 nM) to HMEC-1 cells inhibited constitutive and SFN-induced ARE-linked transcriptional response (Fig. 7D), consistent with acetylation as a likely cause of inactivation of nucleus trapped Nrf2. These studies indicate that Nrf2 loses its transactivational activity with prolonged dwell time in the nucleus where Nrf2 acetylation and nuclear proteasomal degradation are involved.

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