The response of multicellular organisms to stress and maintenance of tissue homeostasis is a common biological problem in all eukaryotes, dictated by both genetic and environmental factors. Highly integrated and stereotypic response patterns are found in many organisms, but the means by which so many diverse pathways, critical for cellular, tissue, and ultimately organismal survival, are coordinated has yet to be elucidated. Here, we show that depletion of the evolutionarily ancient and highly conserved HMGB1 protein inhibits autophagy in human and murine cells. Conversely, inhibition of autophagy limits HMGB1 translocation, suggesting that HMGB1 is central to the regulation of autophagy. Moreover, we have shown that this effect is controlled by ERK1/2 phosphorylation and by interrupting the interaction of Beclin1 with Bcl-2. HMGB1 C106 is important for its translocation between the nucleus and cytoplasm, and oxidized C23/45 is required for the binding to Beclin1. These findings have implications for the temporal and spatial regulation of HMGB1 within cells and tissues, the connection between HMGB1 and autophagic pathways, and the role of HMGB1 as a critical regulator of cell survival.
HMGB1 protein is both a nuclear DNA binding factor and a secreted protein that is critically important for cell death and survival (Tang et al., 2010b
). Its activities are determined by its intracellular localization and posttranslational modifications (including acetylation, ADP-ribosylation, phosphorylation, and thiol oxidation; Scaffidi et al., 2002
; Lotze and Tracey, 2005
; Hoppe et al., 2006
; Kazama et al., 2008
). HMGB1 is released during necrosis as an endogenous damage-associated molecular pattern molecule, or “danger” signal, during cell death (Scaffidi et al., 2002
). Late-stage apoptotic cells can indeed release HMGB1, and oxidation of HMGB1 interferes with its ability to promote immunity (Kazama et al., 2008
). ROS also function as signaling molecules in various pathways regulating both cell survival and cell death. ROS can induce autophagy through several distinct mechanisms involving catalase activation of Atg4 and disturbances in the mETC (Scherz-Shouval et al., 2007
). We have shown here that ROS generated during starvation and rapamycin treatment serve as signaling molecules that initiate autophagy. Importantly, these stimuli caused HMGB1 cytosolic translocation from the nucleus, indicating that the function and localization of HMGB1 necessary to enhance autophagy are different from that observed in apoptosis. Moreover, we have demonstrated that HMGB1 translocation in autophagy is ROS dependent. Indeed, oxidative stress regulates HMGB1 release and subsequent inflammation in recruited macrophages and neutrophils and in ischemic tissues, including hepatocytes (Tang et al., 2007c
; Tsung et al., 2007
These findings ( and ) suggest that HMGB1 is directly involved in the positive regulation and maintenance of autophagy in stressed cells. Autophagy was first discovered as a nonselective pathway for the degradation of intracellular constituents, activated in response to starvation (Maiuri et al., 2007
; Levine and Kroemer, 2008
). It is now clear that selective autophagic degradation of specific organelles and proteins occurs in response to diverse stimuli, varying from survival-promoting removal of pathogens, to degradation of damaged organelles and proteins, to programmed cell survival or cell death (Scherz-Shouval and Elazar, 2007
). We found that targeted deletion of HMGB1 limits starvation-induced autophagy and enhances apoptosis. In the setting of cancer, overexpression of HMGB1 is associated with aberrant survival of many types of cancer, including breast, colon, melanoma, and others (Tang et al., 2010b
). Thus, HMGB1 plays an important role in the regulation of autophagy in response to metabolic stress, oxidative injury, and genomic instability promoting programmed cell survival.
This study demonstrates that HMGB1 promotes and sustains autophagy, and we explored the mechanism by which this occurs. HMGB1 confers pro-autophagic activities, likely by controlling Beclin1–Bcl-2 complex formation. The dissociation of Bcl-2 from Beclin1 is an important mechanism involved in activating autophagy and limiting apoptosis in response to starvation and potentially after other physiological stimuli (Pattingre et al., 2005
). Before this study, the regulation of the interaction between Bcl-2 and Beclin1 by adverse nutrient conditions was not thoroughly explored. We found that HMGB1 disrupts the interaction between Beclin1 and Bcl-2 by competitively binding to Beclin1 after starvation. The oxidation-sensitive C106, but not the vicinal C23 and C45, of HMGB1 regulated cytosolic localization and autophagy. C106S mutants have much higher cytoplasmic levels of HMGB1 and demonstrate enhanced binding to Beclin1, leading to the subsequent dissociation of Bcl-2 from Beclin1. C106S mutants were capable of initiating starvation-induced autophagy as efficiently as wild-type HMGB1, whereas C23S and C45S mutations abolished this effect. Thus, in two independent experiments, C106S mutants behave in a similar fashion to wild-type HMGB1. Meanwhile, C23S and C45S mutants lose their ability to mediate autophagy, as they are unable to bind Beclin1 and, therefore, cannot disrupt Bcl-2–Beclin1 interactions. These findings demonstrate that oxidation of HMGB1 regulates its localization and ability to sustain autophagy (). It thus appears that cytoplasmic translocation of HMGB1 is necessary but may not be sufficient to enhance autophagy.
The antiapoptotic molecule Bcl-2 is involved in the regulation of cell death and survival pathways during apoptosis and autophagy. It was previously reported that the phosphorylation of Bcl-2, likely by kinases of the JNK or ERK signaling pathway, is required for its antiapoptotic activity (Subramanian and Shaha, 2007
; Wei et al., 2008
). Our findings suggest that HMGB1 may also be involved in the regulation of Bcl-2 phosphorylation by the ERK/MAPK pathway because ablation of HMGB1 diminishes starvation-induced phosphorylation of both ERK1/2 and Bcl-2, although the mechanism of this effect is not clear. A recent study has shown that the HMGB1 receptor, receptor for advanced glycation endproducts, directly binds to ERK through a D domain–like docking site and regulates p-ERK1/2 (Ishihara et al., 2003
). It is possible that cytoplasmic HMGB1 interacts with the cytosolic domain of receptor for advanced glycation endproducts and mediates concomitant ERK1/2 and Bcl-2 phosphorylation.
Based on the work reported here and previous findings implicating the nuclear sequestration of HMGB1 in apoptosis, its extracellular release in inflammation (Wang et al., 1999
; Scaffidi et al., 2002
; Kazama et al., 2008
), and its critical role as a universal DNA sensor (Yanai et al., 2009
; Sims et al., 2010
), we propose a more nuanced conceptual model involving HMGB1 and cell death. In this model, ROS generated by cellular stress promote HMGB1 translocation to the cytosol, which induces autophagy, enhancing ERK signaling and disrupting Beclin1–Bcl-2 complex formation (). Thus, endogenous HMGB1 plays a central role in regulating programmed cell death (apoptosis) and programmed cell survival (autophagy). These findings will be instrumental in developing more effective therapies in the context of chronic inflammatory diseases, some of them associated with release of damage-associated molecular proteins (Zhang et al., 2010
), including neurodegenerative disorders, autoimmunity, and cancer.