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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

Evidence against the accumulation of Nrf2 protein during the activation of ARE-linked gene expression. Cellular Nrf2 and Keap1 during the induction of NQO1 expression by 2 μM SFN: (A) Nrf2 (NFE2L2) mRNA, (B) Nrf2 protein, (C) NQO1 mRNA, (D) NQO1 protein (E) Keap1 mRNA and (F) Keap1 protein, and. Key: □---□, control; ■—■, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Examination of short-period variability of Nrf2, Keap1 and NQO1 in the initial 3 h exposure period: (G) Nrf2 protein, (H) Keap1 protein, (I) NQO1 protein, (J) Nrf2 mRNA and (K) NQO1 mRNA. Key: ····, control; —, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human aortic endothelial cells in primary culture: (L) Nrf2 protein, (M) NQO1 mRNA. Key: □---□, control; ■—■, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human BJ fibroblasts in primary culture: (N) Nrf2 protein, (O) NQO1 mRNA. Key: □---□, control; ■—■, +1 μM SFN. For (G–O), data are mean with bars showing the range of two estimates; and for (J, K), data are mean±SD (n=3). Significance: (G–I), autocorrelation function analysis for white noise—p>0.05 for all panels; other panels— *p<0.05, **p<0.01 and ***p<0.001 with respect to unstimulated control (t-test).
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f4: Evidence against the accumulation of Nrf2 protein during the activation of ARE-linked gene expression. Cellular Nrf2 and Keap1 during the induction of NQO1 expression by 2 μM SFN: (A) Nrf2 (NFE2L2) mRNA, (B) Nrf2 protein, (C) NQO1 mRNA, (D) NQO1 protein (E) Keap1 mRNA and (F) Keap1 protein, and. Key: □---□, control; ■—■, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Examination of short-period variability of Nrf2, Keap1 and NQO1 in the initial 3 h exposure period: (G) Nrf2 protein, (H) Keap1 protein, (I) NQO1 protein, (J) Nrf2 mRNA and (K) NQO1 mRNA. Key: ····, control; —, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human aortic endothelial cells in primary culture: (L) Nrf2 protein, (M) NQO1 mRNA. Key: □---□, control; ■—■, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human BJ fibroblasts in primary culture: (N) Nrf2 protein, (O) NQO1 mRNA. Key: □---□, control; ■—■, +1 μM SFN. For (G–O), data are mean with bars showing the range of two estimates; and for (J, K), data are mean±SD (n=3). Significance: (G–I), autocorrelation function analysis for white noise—p>0.05 for all panels; other panels— *p<0.05, **p<0.01 and ***p<0.001 with respect to unstimulated control (t-test).

Mentions: To examine Nrf2 stress response signaling between cytoplasmic and nuclear compartments we studied real-time changes in subcellular localization of Nrf2 in human HMEC-1 (human microvascular endothelial cell line-1) cells in vitro by live cell time-lapse fluorescence microscopy. HMEC-1 cells were transfected to express green fluorescent protein (GFP)-Nrf2 fusion protein. Dynamics of nucleus/cell GFP-Nrf2 fluorescence intensity ratio obtained from time-lapse image sequences revealed that Nrf2 undergoes oscillatory cytoplasm-nucleus translocation. Observation periods were for 400 min (Fig. 2A and Supplementary Video S1; Supplementary Data are available online at www.liebertpub.com/ars). Total cell fluorescence was unchanged over this period—in keeping with unchanged total cellular Nrf2 protein content (see Fig. 4B, L, and N). The fluorescence was quantified by computing the mean fluorescence per pixel in the nucleus and whole cell, ĪNucleus and ĪCell respectively. Average pixel intensities are used because they are less dependent upon cell size. The ratio ĪNucleus/ĪCell showed the periodicity of the translocational oscillations (Fig. 2B). The accumulation of Nrf2 into the nucleus was slow and, after a time delay, its expulsion from the nucleus was relatively rapid. Oscillations for individual cells were asynchronous with those of other cells. The period of oscillation, median (lower–upper quartile), was: 129 (81–175) min (n=44) with amplitude 0.65 (0.35–0.87) arbitrary units (Fig. 2D).


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)

Evidence against the accumulation of Nrf2 protein during the activation of ARE-linked gene expression. Cellular Nrf2 and Keap1 during the induction of NQO1 expression by 2 μM SFN: (A) Nrf2 (NFE2L2) mRNA, (B) Nrf2 protein, (C) NQO1 mRNA, (D) NQO1 protein (E) Keap1 mRNA and (F) Keap1 protein, and. Key: □---□, control; ■—■, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Examination of short-period variability of Nrf2, Keap1 and NQO1 in the initial 3 h exposure period: (G) Nrf2 protein, (H) Keap1 protein, (I) NQO1 protein, (J) Nrf2 mRNA and (K) NQO1 mRNA. Key: ····, control; —, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human aortic endothelial cells in primary culture: (L) Nrf2 protein, (M) NQO1 mRNA. Key: □---□, control; ■—■, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human BJ fibroblasts in primary culture: (N) Nrf2 protein, (O) NQO1 mRNA. Key: □---□, control; ■—■, +1 μM SFN. For (G–O), data are mean with bars showing the range of two estimates; and for (J, K), data are mean±SD (n=3). Significance: (G–I), autocorrelation function analysis for white noise—p>0.05 for all panels; other panels— *p<0.05, **p<0.01 and ***p<0.001 with respect to unstimulated control (t-test).
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f4: Evidence against the accumulation of Nrf2 protein during the activation of ARE-linked gene expression. Cellular Nrf2 and Keap1 during the induction of NQO1 expression by 2 μM SFN: (A) Nrf2 (NFE2L2) mRNA, (B) Nrf2 protein, (C) NQO1 mRNA, (D) NQO1 protein (E) Keap1 mRNA and (F) Keap1 protein, and. Key: □---□, control; ■—■, +2 μM SFN. Data are mean±SD (n=3), normalized to baseline level. Examination of short-period variability of Nrf2, Keap1 and NQO1 in the initial 3 h exposure period: (G) Nrf2 protein, (H) Keap1 protein, (I) NQO1 protein, (J) Nrf2 mRNA and (K) NQO1 mRNA. Key: ····, control; —, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human aortic endothelial cells in primary culture: (L) Nrf2 protein, (M) NQO1 mRNA. Key: □---□, control; ■—■, +2 μM SFN. Cellular Nrf2 during the induction of NQO1 expression by SFN in human BJ fibroblasts in primary culture: (N) Nrf2 protein, (O) NQO1 mRNA. Key: □---□, control; ■—■, +1 μM SFN. For (G–O), data are mean with bars showing the range of two estimates; and for (J, K), data are mean±SD (n=3). Significance: (G–I), autocorrelation function analysis for white noise—p>0.05 for all panels; other panels— *p<0.05, **p<0.01 and ***p<0.001 with respect to unstimulated control (t-test).
Mentions: To examine Nrf2 stress response signaling between cytoplasmic and nuclear compartments we studied real-time changes in subcellular localization of Nrf2 in human HMEC-1 (human microvascular endothelial cell line-1) cells in vitro by live cell time-lapse fluorescence microscopy. HMEC-1 cells were transfected to express green fluorescent protein (GFP)-Nrf2 fusion protein. Dynamics of nucleus/cell GFP-Nrf2 fluorescence intensity ratio obtained from time-lapse image sequences revealed that Nrf2 undergoes oscillatory cytoplasm-nucleus translocation. Observation periods were for 400 min (Fig. 2A and Supplementary Video S1; Supplementary Data are available online at www.liebertpub.com/ars). Total cell fluorescence was unchanged over this period—in keeping with unchanged total cellular Nrf2 protein content (see Fig. 4B, L, and N). The fluorescence was quantified by computing the mean fluorescence per pixel in the nucleus and whole cell, ĪNucleus and ĪCell respectively. Average pixel intensities are used because they are less dependent upon cell size. The ratio ĪNucleus/ĪCell showed the periodicity of the translocational oscillations (Fig. 2B). The accumulation of Nrf2 into the nucleus was slow and, after a time delay, its expulsion from the nucleus was relatively rapid. Oscillations for individual cells were asynchronous with those of other cells. The period of oscillation, median (lower–upper quartile), was: 129 (81–175) min (n=44) with amplitude 0.65 (0.35–0.87) arbitrary units (Fig. 2D).

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