Reactive oxygen species (ROS)–dependent signals are required for HMGB1 translocation and enhanced autophagy

ROS are signaling molecules important in several pathways that regulate both cell survival and cell death. Indeed, many stimuli that induce ROS generation, such as nutrient starvation, mitochondrial toxins, and hypoxia, also induce autophagy (Fig. S2). ROS formation in the mitochondria is a fundamental regulatory event that promotes autophagy (Scherz-Shouval and Elazar, 2007). The major source of endogenous ROS is the mitochondrial electron transport chain (mETC; Scherz-Shouval and Elazar, 2007).

To evaluate the relationship between mitochondrial ROS production and HMGB1 translocation during autophagy, we treated cells with several different mETC inhibitors. Treatment with rotenone (Rot; complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), and antimycin A (complex III inhibitor) increased ROS production, LC3 punctae, and LC3-II expression (Fig. 1, E and F). These mETC inhibitors also induced HMGB1 translocation from the nucleus to the cytosol, as assessed by imaging cytometry and Western blot of subcellular fractions (Fig. 1, E and F). Under physiological conditions, ROS are cleared from cells by the action of the superoxide dismutase (SOD) family members, catalase, or glutathione peroxidase (Scherz-Shouval and Elazar, 2007). As expected, knockdown of SOD1 and SOD2 increased starvation- and rapamycin-induced autophagy and HMGB1 translocation in Panc2.03 cells (Fig. 1, G and H; and Fig. S3). Furthermore, N-acetyl cysteine (NAC), an ROS quencher, dose-dependently inhibited starvation- and rapamycin-induced autophagy and HMGB1 translocation (Fig. 1, G and H; and Fig. S3). Moreover, starvation and rapamycin increased ROS/mitochondrial superoxide levels and decreased SOD activity but did not affect catalase activity or detected levels (Fig. S2). Together, these results are consistent with the notion that ROS signals are required for HMGB1 translocation and sustained autophagy.

Lack of HMGB1 limits autophagy

To assess the role of HMGB1 in autophagy, we first evaluated autophagic flux in wild-type and Hmgb1^−/− MEFs. Hmgb1^−/− MEFs had markedly diminished LC3-GFP punctae, endogenous LC3 punctae formation, and LC3-II expression in cells after treatment with several autophagic stimuli, including H₂O₂, rapamycin, and starvation (Fig. 2, A–C). Treatment with the lysosomal protease inhibitors pepstatin or E64D (Mizushima and Yoshimori, 2007) induced a further increase in LC3-GFP punctae and LC3-II expression in wild-type, but not Hmgb1^−/−, MEFs (Fig. 2 C). Similarly, knockdown of HMGB1 in HCT116 and Panc2.03 cells also decreased accumulation of LC3 punctae and expression of LC3-II (Fig. S4). Moreover, up-regulation of HMGB1 expression in Hmgb1^−/− MEFs after gene transfection restored LC3 punctae formation (Fig. 2 D). Inhibition of autophagy leads to an increase in the size and number of p62 bodies and p62 protein levels (Bjørkøy et al., 2005). Loss of HMGB1 increases the size and number of p62 bodies and p62 protein levels (Fig. 2 E), indicating that its degradation is dependent on HMGB1-mediated autophagy. Ultrastructural analysis revealed that Hmgb1^−/− MEFs exhibited fewer type I autophagosomes and type II autophagolysosomes when compared with wild-type MEFs (Fig. 2 F). These results indicate that HMGB1 is indeed an important factor that regulates autophagy.

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Figure 2.

Depletion of HMGB1 inhibits autophagy. (A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1^−/− and HMGB1^+/+ MEFs were treated with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1^+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1^−/− and HMGB1^+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A. (C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1^+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units. (D) Up-regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1^−/− MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3. Non, nontransfected. (E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1^+/+ group; n = 3. Representative images are depicted (left). (F) Ultrastructural features in Hmgb1^−/− and Hmgb1^+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a–e point to autophagosomes and autolysosomes). Data are means ± SEM.

Under normal conditions, 5–10% of HMGB1 protein is located in the cytosol, with the specific quantity dependent on the cell type (Fig. 1 A and Fig. 3 A). Moreover, autophagic stimuli promote translocation of HMGB1 to the cytosol (Fig. 1 A and Fig. 3 A). To assess the role of cytoplasmic HMGB1 in the setting of autophagy, we created cytoplasts (anucleate cells). In response to starvation (HBSS), Hmgb1^+/+ MEF cytoplasts were still able to accumulate LC3 punctae (Fig. 3, A and B; Tasdemir et al., 2008). In contrast, Hmgb1^−/− MEF cytoplasts demonstrated a lower level of LC3 punctae than HMGB1^+/+ (Fig. 3, A and B), indicating that cytoplasmic HMGB1 was required for starvation-stimulated autophagy.

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Figure 3.

Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1^−/− and Hmgb1^+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1^−/− and Hmgb1^+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.

Both starvation and rapamycin treatment enhance autophagy and induce apoptosis in some cell lines (Maiuri et al., 2007). We therefore evaluated the relationship between HMGB1 and apoptosis under these conditions. Increased apoptosis was observed in Hmgb1^−/− MEFs when compared with wild-type cells by flow cytometry using an annexin V stain, by finding increased cleaved poly(ADP-ribose) polymerase (PARP), and by increased caspase-3 activation (Fig. S5). Collectively, these data suggest that HMGB1 plays a major role in promoting autophagy and limiting apoptosis.

Depletion of HMGB1 promotes persistent Beclin1–Bcl-2 interaction

An important molecular event observed during autophagy is the disassociation of the Bcl-2–Beclin1 complex (Pattingre et al., 2005). The Bcl-2 family of antiapoptotic proteins regulates Beclin1-dependent autophagy (Pattingre et al., 2005). Thus, we determined the effect of HMGB1 on the interaction between Bcl-2 and Beclin1. The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitors PD98059 and U0126 inhibited starvation-induced phosphorylation of ERK (p-ERK1/2) and Bcl-2 (Fig. 4 A). Moreover, knockout of HMGB1, as well as addition of the MEK/ERK inhibitor U0126, blocked the dissociation of Bcl-2–Beclin1 in the setting of enhanced autophagy (Fig. 4, B and F), suggesting that ERK1/2-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. When present, endogenous HMGB1 demonstrated an interaction with Beclin1 by coimmunoprecipitation (IP [co-IP]) assay in MEFs, HCT116, and Panc02 cells (Fig. 4, B, C, and E). Knockdown of Beclin1 inhibited the interaction between Bcl-2 and HMGB1 in untreated HMGB1 wild-type MEFs (Fig. 4, B and D), suggesting that HMGB1 interaction with Bcl-2 is directly dependent on Beclin1. Thus, via binding to Beclin1 and regulating the phosphorylation of Bcl-2 in cells after starvation, endogenous HMGB1 inhibits the interaction between Beclin1 and Bcl-2.

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Figure 4.

Absence of HMGB1 sustains Beclin1–Bcl-2 interactions. (A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1^−/− and HMGB1^+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting. (B) Knockout of HMGB1 limits the disassociation of the Bcl-2–Beclin1 complex during treatment with autophagic stimuli. HMGB1^−/− and HMGB1^+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting. (C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting. (D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control. (E and F) Quantitative data demonstrating the interaction between HMGB1–Beclin1 and Beclin1–HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A–D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.

Cell culture

Panc2.03, Panc02, HCT116, and Hmgb1^−/− and Hmgb1^+/+ immortalized MEF (Scaffidi et al., 2002) cells were cultured in RPMI 1640, DME, or McCoy’s 5a medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, and antibiotic–antifungal mix (10,000 units/ml penicillin and 10,000 µg/ml streptomycin; Invitrogen) in a humidified incubator with 5% CO₂.

Gene transfection and RNAi

HMGB1-GFP or mutant (C23S, C45S, or C106S; Hoppe et al., 2006) or pUNO1-HMGB1 (InvivoGen) expression vectors were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Constructs were sequenced and confirmed in the University of Pittsburgh core sequencing laboratories. Human SOD1/SOD2-siRNA, mouse Beclin1–Bcl-2 siRNA (Santa Cruz Biotechnology, Inc.), mouse Beclin1 short hairpin RNA (shRNA), human HMGB1 shRNA, and mouse ATG5 shRNA (Sigma-Aldrich) were transfected into cells using Lipofectamine RNAiMAX reagent (for siRNA) or Lipofectamine 2000 reagent (for shRNA) according to the manufacturer’s instructions (Invitrogen). After gene transfection and RNAi for 48 h, the medium over the cells was changed before subsequent treatments.

Western blotting

Whole-cell lysates were resolved on 4–12% Criterion XT Bis-Tris gels (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane as previously described (Tang et al., 2007b). After blocking, the membrane was incubated for 2 h at 25°C or overnight at 4°C with various primary antibodies. After incubation with peroxidase-conjugated secondary antibodies for 1 h at 25°C, the signals were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) according to the manufacturer’s instruction. The relative band intensity was quantified using the Gel-pro Analyzer software (Media Cybernetics).

Measurement of intracellular ROS and mitochondrial superoxide

Cells were seeded in 96-well plates and cultured in the presence of a given stimuli for the indicated time. ROS were assessed with cell-permeable dye CM-H2DCFDA (Invitrogen) and mitochondrial superoxide using MitoSox (Invitrogen) as described previously (Kang et al., 2010b). Analysis of signal intensity was performed using a fluorescent plate reader.

IP analysis

Cells were lysed at 4°C in ice-cold radioimmunoprecipitation assay lysis buffer (Millipore), and cell lysates were cleared by a brief centrifugation for 10 min at 12,000 g. Concentrations of proteins in the supernatant were determined by bicinchoninic acid assay. Before IP, samples containing equal amounts of proteins were precleared with protein A or protein G agarose/Sepharose at 4°C for 3 h and subsequently incubated with 2–5 µg/ml various irrelevant IgG or specific antibodies in the presence of protein A or G agarose/Sepharose beads for 2 h or overnight at 4°C with gentle shaking (Tang et al., 2007a,b). After incubation, agarose/Sepharose beads were washed extensively with PBS, and proteins were eluted by boiling in 2× SDS sample buffer before SDS-PAGE electrophoresis. As a control, a portion of the lysate was treated with 50 mM DTT to reduce sample as described previously (Anathy et al., 2009).

Analysis of HMGB1 translocation and LC3 punctae formation by imaging cytometry

Cells were seeded in 96-well plates, cultured in the presence of various stimuli for given times, and then were fixed with 3% paraformaldehyde and stained with HMGB1 or LC3 antibody. Secondary antibodies were goat IgG conjugated either with Alexa Fluor 488 or Alexa Fluor 647 fluorochromes. Nuclear morphology was visualized with the fluorescent dye Hoechst 33342 (Sigma-Aldrich). Imaging data were collected with an imaging cytometer (ArrayScan HCS 4.0; Cellomics) with a 20x objective at 25°C. The ArrayScan is an automated fluorescent imaging microscope that collects information about the spatial distribution of fluorescently labeled components in cells placed in 96-well microtiter plates. The Spot Detector BioApplication (Thermo Fisher Scientific) was used to acquire and analyze the images after optimization. Images of 500–1,000 cells for each treatment group were analyzed to obtain the mean nuclear and cytosolic HMGB1 intensity and LC3 fluorescence punctae number per cell (Tang et al., 2009).

Autophagy assays

All assays for endogenous LC3 punctae were performed by imaging cytometry as previously described (Kang et al., 2010a). Formation of autophagic vesicles was further monitored by transient expression of GFP-LC3 (gifts of X.-M. Yin, University of Pittsburgh, Pittsburgh, PA) aggregation in cell lines. The percentage of cells with GFP-LC3 punctae was determined by counting the number of positively staining cells from 100 randomly chosen cells from three separate experiments. Autophagic flux assays were performed by Western blotting for LC3-I/II formation after starvation in the presence or absence of lysosomal protease inhibitors (E64d/pepstatin A) as recommended (Mizushima and Yoshimori, 2007).

Confocal microscopy analysis of p62 and colocalization of LAMP2/LC3 were performed. Images were collected using a laser-scanning confocal microscope (Fluoview FV-1000; Olympus) using a 60x Plan Apo/1.45 oil immersion objective at 25°C and were captured and analyzed by Fluoview software (FV10-ASW 1.6; Olympus). Images were subsequently analyzed for the level and colocalization by Image-Pro Plus 5.1 software (Media Cybernetics).

Transmission electron microscopy assessment of autophagosome and autophagolysosomes was performed as previously described (Shao et al., 2004). In brief, cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, followed by postfixation for 6 h in 1% O_sO₄. After dehydration with graded alcohols, the samples were embedded in epoxy resin (epon). Then, thin sections (70 nm) were cut with a microtome (Ultracut R; Leica), mounted on copper grids and poststained with 2% uranyl acetate and 1% lead citrate, dried, and analyzed using a transmission electron microscope at 25°C (100CX; JEOL, Inc.). Thick sections were cut (300 nm) and stained with 1% toluidine blue. Images were acquired digitally from a randomly selected pool of 10–15 fields under each condition.

Apoptosis assays

Apoptosis in cells was assessed using the FITC Annexin V Apoptosis Detection kit (BD; annexin V–FITC, propidium iodide (PI) solution, and annexin V binding buffer). This assay involves staining cells with annexin V–FITC (a phospholipid-binding protein that binds to disrupted cell membranes) in combination with PI (a vital dye that binds to DNA penetrating into apoptotic cells). Flow cytometric analysis (FACS) was performed to determine the percentage of cells that were undergoing apoptosis (annexin V⁺/PI⁻). Cleaved PARP and cleaved caspase-3 were measured by Western blotting analysis.

Enucleation by centrifugation

HMGB1^−/− and HMGB1^+/+ MEFs grown on collagen-coated minicoverslips were enucleated as described previously, with minor modifications (Goldman et al., 1973; Miller and Ruddle, 1974). To enucleate the cells, the minicoverslips were inverted (cell side down) and placed into the bottom of 1.5-ml Eppendorf tubes containing 10 µg/ml cytochalasin B (Sigma-Aldrich) in medium. After centrifugation at 12,000 g for 60 min, the minicoverslips were removed from the tubes and placed cell side up into a 6-well plate containing 2 ml of medium without cytochalasin B for subsequent studies and staining for autophagy.

Statistical analysis

Data are expressed as means ± SEM of three independent experiments performed in triplicate. One-way analysis of variance was used for comparison among the different groups by SPSS 12.0. When the analysis of variance was significant, post-hoc testing of differences between groups was performed using Fisher’s least significant difference test. A p-value <0.05 was considered significant; P < 0.005 and P < 0.0005 were reported where applicable.

Thoughtful discussions and review of this work with Timothy Billiar and Sarah Berman at the University of Pittsburgh and external colleagues Guido Kroemer, Douglas Green, Matthew Albert, Eva Szegezdi, and Beth Levine were much appreciated. We also thank the reviewers for constructive suggestions.

This project was funded by the National Institutes of Health (an Integrating NK and DC into Cancer Therapy grant [1 P01 CA 101944-04] from the National Cancer Institute to M.T. Lotze) and a grant from Associazione Italiana Ricerca sul Cancro (to M.E. Bianchi).