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High mobility group box 1 (HMGB1) activates an autophagic response to oxidative stress - PubMed

  • ️Sat Jan 01 2011

High mobility group box 1 (HMGB1) activates an autophagic response to oxidative stress

Daolin Tang et al. Antioxid Redox Signal. 2011.

Abstract

Aims: Autophagy, the process by which cells break down spent biochemical and damaged components, plays an important role in cell survival following stress. High mobility group box 1 (HMGB1) regulates autophagy in response to oxidative stress.

Results: Exogenous hydrogen peroxide (H(2)O(2)) treatment or knockdown of the major superoxide scavenger enzyme, superoxide dismutase 1 (SOD1), by small interfering RNA (siRNA) increases autophagy in mouse and human cell lines. Addition of either SOD1 siRNA or H(2)O(2) promotes cytosolic HMGB1 expression and extracellular release. Importantly, inhibition of HMGB1 release or loss of HMGB1 decreases the number of autophagolysosomes and autophagic flux under oxidative stress in vivo and in vitro.

Innovation: HMGB1 release may be a common mediator of response to oxidative stress.

Conclusion: HMGB1 is important for oxidative stress-mediated autophagy and serves as a new target for the treatment of stress-associated disorders.

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Figures

FIG. 1.
FIG. 1.

Knockdown of SOD1 by siRNA induces autophagy. (A) Time-dependent effects of SOD1 downregulation. MEFs were transfected with SOD siRNA or control siRNA. After transfection for the indicated time, total protein extracts were used for Western blot analysis. Actin was used as a loading control. Immunoblot shown is representative of three experiments with similar results. (B, C) Confocal microscopic analysis of LC3 (green) and p62 (red) using specific antibodies after transfection with SOD1 siRNA and control siRNA for 48 h in MEFs. Images are representative of 10 random fields. Bar=30 μm. (To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars

).

FIG. 2.
FIG. 2.

The antioxidant NAC inhibits oxidative stress-induced autophagy. (A, B) NAC inhibits SOD1 siRNA-induced autophagy. MEFs were transfected with SOD siRNA or control siRNA for 48 h, and then treated with NAC (50 mM) for 12 h. The total protein extracts were used for Western blot analysis. Data are representative of three experiments with similar results (A). In parallel, LC3 punctae formation (green) and p62 (red) were assayed by confocal microscopic analysis. Images are representative of 10 random fields. Bar=30 μm (B). (C, D) NAC inhibits H2O2-induced autophagy. MEFs were treated with H2O2 (0.05 mM) for 12 h with or without NAC (50 mM). The total protein extracts were used for Western blot analysis. Data are representative of three experiments with similar results (C). In parallel, LC3 punctae formation (green) and p62 (red) were assayed by confocal microscopy analysis. Images are representative of 10 random fields. Bar=30 μm (D). (To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars

).

FIG. 3.
FIG. 3.

Knockdown of SOD1 or H2O2 increases cytoplasmic translocation and release of HMGB1. (A) Confocal microscopic analysis of HMGB1 (red) using specific antibodies following transfection with SOD1 siRNA or control siRNA for 48 h or treatment with H2O2 (0.05 mM) for 12 h with or without NAC (50 mM) in MEFs. Bar=30 μm. Relative quantitative analysis of extranuclear HMGB1 fluorescent intensity shown as means±SD from 10 random fields (*p<0.05. One-way ANOVA, followed by LSD). AU: Arbitrary Units. Representative images of HMGB1 overlaid on the differential interference contrast (DIC) images are shown in the lower panel. (B) Western blot analysis of HMGB1 levels in isolated mitochondria (Mit) with or without H2O2 (0.05 mM) for 12 h in MEFs. Untreated whole cell lysate was used as a positive control (Ctrl) for nonmitochondrial protein. To confirm that these were the appropriate fractions, the Western blots were probed for COX IV as a mitochondrial marker, tubulin as a cytoplasmic marker, and histone H3 as a nuclear marker. (C) Conditions as indicated in (A), the level of HMGB1 and LDH released into the cell culture medium was assayed by Western blot analysis. Relative quantitative analysis of HMGB1 band density shown in top panel (AU: arbitrary units). (D) Analysis of HMGB1 release by ELISA in the presence or absence of 3-methyladenine (“3-MA”, 10 mM) after SOD1 siRNA or control siRNA for 48 h or treatment with H2O2 (0.05 mM) for 12 h. In parallel, the cell viability was analyzed using a CCK-8 kit (n=3, *p<0.05). (E) Analysis of HMGB1 translocation by confocal microscopy in Atg5+/+ and Atg5−/− MEFs after treatment with H2O2 (0.05 mM) for 12 h. Representative images of HMGB1 location (red) is shown in left panel. Bar=30 μm. In parallel, HMGB1 release was assessed by ELISA (n=3, *p<0.05). (F) MEFs were treated with H2O2 (0.25 mM) in the presence or absence of the PARP inhibitor DHIQ (300 μM). At 12 h following treatment, the cell viability was analyzed using a CCK-8 kit (n=3, *p<0.05). (G) LC3 punctae formation was determined by confocal microscopic analysis in the presence or absence of HMGB1-neutralizing antibody (10 μg/ml) after SOD1 siRNA or control siRNA for 48 h or treatment with H2O2 (0.05 mM) for 12 h (n=10 random fields, *p<0.05). (H) Loss of RAGE expression increases H2O2-induced oxidative cytotoxicity. HMGB1+/+ and HMGB1−/− MEFs were treated with H2O2 at indicated dose for 24 h and then the cell viability was analyzed (n=3, *p<0.05 versus HMGB1−/− MEFs). (To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars

).

FIG. 4.
FIG. 4.

Autophagy induced by oxidative stress is mediated by HMGB1. (A) Western blot analysis of indicated protein levels in HMGB1+/+ and HMGB1−/− MEFs after transfection with SOD1 siRNA or control siRNA for 48 h or treatment with H2O2 (0.05 mM) for 12 h. Immunoblot shown is representative of three experiments with similar results. (B, C) In parallel, LC3 punctae formation was assayed by confocal microscopic analysis in MEFs or HMGB1 knockdown HCT116 and Panc02 cells (*p<0.05. One-way ANOVA, followed by LSD). KD: HMGB1 knockdown, WT: HMGB1 wild type. Images are representative of 10 random fields. Bar=30 μm (MEFs); Bar=15 μm (HCT116 and Panc02). (D) Co-localization of LC3 (green)/LAMP2 (red) was assayed by confocal microscopic analysis (*p<0.05. One-way ANOVA, followed by LSD) using the conditions indicated in (A). Images are representative of 10 random fields. Baf A: Bafilomycin A1 (100 nM); Bar=30 μm. In parallel, LC3 expression was assayed by Western blot analysis (top panel). (E) HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1 cDNA as indicated and then were treated with H2O2 (0.05 mM) for 12 h. The LC3 punctae formation was assayed by confocal microscopic analysis (*p<0.05. One-way ANOVA, followed by LSD, n=10 random fields). (To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars

).

FIG. 5.
FIG. 5.

Expression of HMGB1 mediates chemoresistance in vivo by reducing apoptosis and enhancing autophagic flux during oxidative stress. (A) HMGB1 knockdown tumor cells are more sensitive to gemcitabine in vivo. C57/BLl6 mice were inoculated with 106 Panc02 tumor cell following transfection of control or HMGB1-specific shRNA and treated with gemcitabine (GEM, 15 mg/kg, twice/week) or PBS beginning on day 10. Tumors were measured twice weekly, and volumes were calculated for 42 day (n=5 mice/group and expressed as mean±SD, *p<0.05). (B) On day 42, HMGB1 expression, apoptosis (TUNEL), autophagy (LC3), and oxidative stress (HNE) in tumor samples were assayed by immunofluorescence. (To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars

).

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