HMGB1 and autophagy

TANG Dao-lin

The word"autophagy"is derived from the Greek roots"auto"and"phagy"which mean"self"and"to eat"，respectively.As a hot topic and rapidly expanding field in biology and medicine，autophagy is a process by which cytoplasmic components，including soluble macromolecules(such as nucleic acids，proteins，carbohydrates and lipids)and organelles(such as mitochondria，peroxisomes and endoplasmic reticulum)are degraded by lysosomes.There are at least three re cognized types of autophagy including chaperone-mediated autophagy，microautophagy，and macroautophagy［1］.Chaperonemediated autophagy involves direct translocation of the unfolded substrate pentapeptide KFERQ across the lysosome membrane through the action of a cytosolic and lysosomal chaperone

heat shock cognate protein of 70000(Hsc70)，and the integral membrane receptor lysosome-associated membrane protein type 2A(LAMP-2A).Microautophagy refers to the sequestration of cytosolic components directly by lysosomes through invaginations within their limiting membrane.In the case of macroautophagy(hereafter autophagy)，

the cargo is sequestered within a unique double membrane cytosolic vesicle，an autophagosome.The autophagosome is formed by expansion of the phagophore.The autophagosome undergoes fusion with a lysosome to form an autolysosome，in which the sequestered material is degraded.Autophagy，an intrinsically nonselective process，can also target selective cargo for degradation through mitophagy，lipophagy，ribophagy and xenophagy.Compared to cell apoptosis，autophagy is a genetically programmed，evolutionarily conserved cell survival process that degrades longlived cellular proteins and organelles.Autophagy dysfunction is implicated in certain human diseases，including infections，cancer，neurodegeneration，aging，and heart disease［2］.

1 Dual roles of high-mobility group box 1(HMGB1):A DNA binding and extracellular signaling factor

The HMGB1 protein is highly conserved through evolution，and is 99%identical in all mammals.It was first identified as one of a group of chromatin-associated proteins with high acidic and basic amino acid content.HMGB1，composed of the A box(aa 1-79)，B box(aa 89-163)and C tail(aa 186-215)domains，is a redoxsensitive protein［3］.There are three cysteines at positions 23，45 and 106(C23，C45，and C106，respectively).C23 and C45 readily form an intra-molecular disulfide bridge，whereas C106 remains in a reduced form.HMGB1 is a highly conserved nuclear protein，acting as a chromatin-binding factor that bends DNA and promotes the access to transcriptional protein assemblies on specific DNA targets.In addition to its nuclear role，HMGB1 functions as a damage associated molecular pattern(DAMP)molecule，promoting inflammation，cell proliferation，cellular differentiation，survival and migration［4］.HMGB1 is released in two different ways:passively released from necrotic/injured cells and actively secreted by inflammatory cells.Once released，HMGB1 binds with a high affinity to several receptors，including the receptor for advanced glycation end(RAGE)products，Toll-like receptors(TLR)-2，TLR-4，TLR-9，and，as a negative signaling molecule，CD24，mediating the response to infection and immunity.HMGB1 has been implicated in disease states，including sepsis［5］，ischemia-reperfusion，arthritis，meningitis，neurodegeneration，aging and cancer.Unlike other DAMPs，HMGB1 has unique roles in cancer.HMGB1 expression is higher in tumor cells than in normal surrounding epithelia in a large variety of human neoplasms，including lymphoma，melanoma and cancers of the breast，cervix，colon，liver，and lung.The overexpression of HMGB1 is associated with each of the hallmarks of cancer，including unlimited replicative potential，angiogenesis，evasion of cell apoptosis，self-sufficiency in growth signals，insensitivity to inhibitors of growth，inflammation，tissue invasion and metastasis，and enhancement of inflammation［6］.

2 HMGB1 regulates starvation and tumor therapy-induced autophagy

Although substantial information exists about HMGB1 in the setting of apoptosis and necrosis，the role of HMGB1 in autophagy was previously uncharacterized.Our recent study presented the first evidence that HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits apoptotic cell death in normal and cancer cells［7－10］.Notably，we demonstrated that endogenous and exogenous HMGB1 are novel and important regulators of autophagy［7－8，10－15］.Deletion，depletion，or inhibition of HMGB1 in mouse embryonic fibroblasts or tumor cells markedly diminishes microtubule-associated protein light chain 3(LC3)punctae and autophagosome formation and is associated with the increased expression of p62/SQSTM1 in response to classic autophagic stimuli.Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1，and orients Beclin 1 to autophagosomes.Additionally，HMGB1 may be involved in the regulation of Bcl-2 phosphorylation through the extracellular signal regulated kinase(ERK)/MAPK pathway，as ablation of HMGB1 diminishes starvation-induced phosphorylation of both ERK1/2 and Bcl-2.Moreover，the intra-molecular disulfide bridge(C23/45)within HMGB1 is required for binding to Beclin 1 and sustaining autophagy.Extracellular reduced HMGB1 binds the receptor for advanced glycation end products(RAGE)，which inhibits the mammalian target of rapamycin(mTOR)and promotes the formation of the Beclin 1-classⅢ phosphatidylinositol 3-kinase(PI3KC3)complex.This subsequently promotes pancreatic，colon，and leukemia tumor cell resistance to chemotherapeutic agents and ionizing radiation［10，12，15－16］.There is a direct molecular interaction between HMGB1 and p53 in colorectal cancer to regulate apoptosis and autophagy［8］.Loss of p53 increases cytosolic HMGB1，leading to increased binding to Beclin 1，thereby promoting autophagy and decreasing apoptosis.In contrast，loss of HMGB1 increases cytosolic p53 and apoptosis and decreases autophagy.Knockdown or inhibition of HMGB1 decreases the level of autophagy and increases the sensitivity of tumor cells to drug therapy.

3 Metabolic regulation by HMGB1-mediated mitophagy

Mitochondria play a primary role in bioenergetics in most eukaryotic cells.Dysfunctional mitochondria with futile adenosine triphosphate(ATP)hydrolysis generate increased oxidative stress.Furthermore，extensive mitochondrial damage may lead to the dissipation of the membrane potential across the inner membrane and induction of cell death through the release of pro-apoptotic proteins.Mitochondrial autophagy，termed mitophagy，prevents the release of pro-apoptotic proteins from damaged mitochondria and maintains mitochondrial quality and energy balance［17］.Recently，we demonstrated that nuclear HMGB1 regulates mitochondrial biogenesis and morphology through regulation of autophagy and mitophagy［14，18］.ATP is produced by cellular respiration during glycolysis and/or oxidative phosphorylation(OXPHOS)and utilized by enzymes and structural proteins in many cellular processes.Knockout or knockdown of HMGB1 in fibroblasts and cancer cells significantly inhibits both OXPHOS and glycolysis，and ATP production is decreased in HMGB1-deficient cells.Moreover，the expression of the HMGB1 gene in HMGB1-deficient cells restores OXPHOS and glycolysis，suggesting that HMGB1 specifically regulates cellular respiration.

Morphological analysis revealed that mitochondria，which normally exhibit long and tubular morphology in HMGB1+/+mouse embryonic fibroblasts(MEFs)，become dramatically shorter and rounder in HMGB1－/－MEFs.We also demonstrated that heat shock protein beta-1(HSPB1/HSP27)is the downstream mediator of this effect.Disruption of the HSPB1 gene in embryonic fibroblasts with wild-type HMGB1 recapitulates the mitochondrial fragmentation，deficits in mitochondrial respiration，and ATP synthesis observed with targeted deletion of HMGB1.Forced expression of HSPB1 reverses this phenotype in HMGB1 knockout cells.Moreover，the PTEN-induced kinase 1(Pink1)-Parkin mitophagy pathway is required for HMGB1/HSPB1-mediated mitophagy.Thus，the induction of autophagy/mitophagy by HMGB1 is critical for mitochondrial homeostasis and fine-tuning of cellular energetic balance.In addition，RAGE is an important inflammatory mediator that modulates the crosstalk between pro-survival pathways:IL-6/pSTAT3 and autophagy in pancreatic ductal adenocarcinoma tumor cells，and contributes to early pancreatic intraepithelial neoplasia formation［13］.

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